Organophosphorus (OP) compounds are found in household pest control products, plastics, and petroleum. Due to the neurotoxic nature of OP compounds, exposure can cause both acute and delayed symptoms, including organophosphate-induced delayed neuropathy (OPIDN). This syndrome is characterized by Wallerian-like degeneration of nerves in the central and peripheral nervous system after exposure to neuropathic OP compounds. There are many questions surrounding the mechanisms of the onset of OPIDN, including possible alterations in proteins associated with neuronal maintenance and repair. This dissertation investigated the changes in levels of neurotrophins in vivo and how in vitro levels of neurotrophin receptors and their downstream signaling cascades are affected after exposure to OP compounds. We also characterized the molecular weight of a soluble factor responsible for inducing neurite outgrowth in vitro after in vivo exposure to a neuropathic OP compound. We evaluated in vivo endpoints using enzyme-linked immunosorbant assays. Results indicated that nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are found in chicken spinal cord but do not increase as a result of exposure to neuropathic OP compounds. This study also noted that NGF, BDNF, and NT-3 concentrations were not altered after exposure to a non-neuropathic OP compound. We evaluated in vitro endpoints using Western blots, ultrafiltration, and digital morphometry. These studies revealed that activated forms of high-affinity and low-affinity neurotrophin receptors are present after OP compound exposure, that the ratio of these two receptors to each other is stable after OP compound exposure, and that the activated form of the low-affinity receptor, which can lead to apoptosis, was present in greater levels than the activated form of the high-affinity receptor. Furthermore, OP compound exposure resulted in time-dependent changes of protein levels central to the mitogen-activated kinase and phosopholipase C-gamma intracellular pathways. Changes in a third pathway, the protein kinase C pathway, were dependent on the concentration and type of OP compound. Finally, in vitro neurite length was not affected by the type of OP compound administered in vivo or when a whole protein fraction was separated by molecular weight. This research has revealed in vivo consequences and early effects on intracellular protein and activated neurotrophin receptor levels after OP compound exposure. These early effects may contribute to the delayed development of neurotoxic effects associated with OP compound exposure. / Ph. D.

A doenÃa de Parkinson (DP) à caracterizada pela degeneraÃÃo nigroestriatal, com depleÃÃo dopaminÃrgica, alteraÃÃes inflamatÃrias e oxidativas cerebrais levando a prejuÃzo no controle do movimento e coordenaÃÃo. Trabalhos recentes mostram que a atividade fÃsica em esteira pode ser benÃfica para estes pacientes, mas hà poucas evidÃncias avaliando os parÃmetros sanguÃneos relacionados, como estresse oxidativo e nÃveis de neurotrofinas. Assim, o presente estudo objetivou avaliar o impacto do treinamento da marcha atravÃs da esteira ergomÃtrica na capacidade funcional e marcadores de estresse oxidativo bem como os nÃveis de neurotrofinas em pacientes com DP. Tratou-se de ensaio clÃnico controlado, aberto e aleatorizado, de agosto de 2013 a fevereiro de 2014. IncluÃdos 22 indivÃduos com DP estÃgio II e/ou III, na escala de Hoehn e Yahr, acima de 40 anos, e que nÃo apresentaram quadro de demÃncia pelo Mini Teste do Estado Mental (escore> 21). ExcluÃdos os que apresentaram sinais de piora clÃnica apÃs o inicio do estudo com aumento no tremor Ãs atividades e com duas faltas consecutivas ao tratamento proposto ou trÃs no tempo total deste. Os pacientes foram aleatorizados em dois grupos: Grupo Controle (GC) e Grupo de IntervenÃÃo (GI). As avaliaÃÃes relacionadas à capacidade funcional (qualidade de vida, anÃlise estÃtica e dinÃmica da marcha) e parÃmetros sanguÃneos como peroxidaÃÃo lipÃdica (TBARS), glutationa reduzida (GSH) e fator neurotrÃfico derivado do cÃrebro (BDNF) foram realizadas antes e apÃs as oito semanas de intervenÃÃo. O GI iniciou o protocolo individualmente, com duas intervenÃÃes semanais na esteira ergomÃtrica, utilizando 80% da velocidade mÃxima encontrada no protocolo de Harbor, num total de 16 atendimentos. Jà o GC manteve-se somente com o tratamento medicamento previsto. Resultados foram avaliados pelo programa estatÃstico SPSS versÃo 17.0 e expressos como mÃdia  desvio padrÃo, sendo estatisticamente significante valores de p< 0,05. Os dados demogrÃficos dos grupos foram homogÃneos quanto Ãs variÃveis idade, gÃnero, altura, peso, tempo de doenÃa, teste mini mental e teste da escala de depressÃo. A qualidade de vida nÃo mostrou diferenÃas no coeficiente fÃsico sumarizado, entretanto no coeficiente mental sumarizado foi observado diferenÃa entre os grupos no perÃodo pÃs-intervenÃÃo (p=0,03), o SF-36 considera QV acima de 50 pontos. NÃo foram encontradas diferenÃas na superfÃcie do pà (P) direito (D) e esquerdo (E), nem na distÃncia dos pÃs em relaÃÃo ao barocentro, tambÃm nÃo houve diferenÃa na pressÃo mÃdia e mÃxima dos PD e PE, em ambos os grupos. Com relaÃÃo a variÃvel superfÃcie, viu-se diferenÃa no perÃodo prÃ-intervenÃÃo no PE (p=0,001). No perÃodo pÃs-intervenÃÃo houve diferenÃa na superfÃcie do PD (p=0,001), na oscilaÃÃo Ãntero-posterior do PE (0,01) e na oscilaÃÃo lÃtero-lateral do PD (p=0,01). Na velocidade da marcha foi encontrado, antes da intervenÃÃo, diferenÃa na velocidade mÃdia do PD (p=0,04). No GI, viu-se associaÃÃo forte entre BDNF e GSH com valores de significÃncia estatÃstica (r= 0,8; p=0,001), assim como entre BDNF e prÃtica de atividade fÃsica (r= 0,7; p=0,03). Conclui-se que a caminhada controlada na esteira melhora o equilÃbrio estÃtico, qualidade de vida e os nÃveis plasmÃticos de GSH em pacientes com DP. / Parkinson disease (PD) is characterized by nigrostriatal degeneration with consequent depletion of dopamine content in the striatum as well as brain inflammatory and oxidative alterations leading to movement and coordination impairment. Recent studies showed that physical exercise in the treadmill is benefic for PD patients although there is a lack in the literature of plasma alterations in oxidative stress parameters and neurotrophins in PD patients submitted to physical exercise. Based on this, the present study had the goal to evaluate the impact of controlled treadmill walking in the functional capacity and plasma levels of oxidative stress parameters and brain derived neurotrophic factor (BDNF) of PD patients. To do this, participants from both sexes were submitted to an open and randomized trial from august 2013 to February 2014. The participants were randomized in two groups control (CG) and intervention group (IG). The IG comprised PD patients in stage II and/or III, based on the Hoehn and Yahr scale. These patients were 40 years old or above and did not present dementia as evaluated by Mini-mental state examination (score> 21). Patients who presented signals of clinical worsening, such as increased tremor during activities, or presented two consecutive absences during the treatment or a total of three absences were excluded from the study. The evaluation related to the functional capacity (e.g. quality of life, static analyses and dynamic gait) and plasma parameters (lipid peroxidation, reduced glutathione (GSH) and BDNF were performed before and immediately after eight weeks of intervention. The IG group started the protocol individually with two weekly interventions in the treadmill with 80 % of maximum velocity based on the Harbor protocol in a total of 16 interventions. The CG was maintained only in drug treatment. The results were evaluated using the SPSS software version 17.0 considering differences when p<005. The demographic data were homogeneous based on the variables age, sex, weight, time of disease, mini-mental and depression tests. Quality of life did not present significant differences in the physical coefficient summarized, however in the mental coefficient summarized there was a difference between groups in the post intervention period (p= 0.03). There were no differences in the surface of right (RF) and left feet (LF) neither in the distance of the feet in relation to the barocenter. There was no alteration in the medium and maximal pressure of RF and LF. In relation to the variable surface, a significant difference in the period pre-intervention in the LF was observed (p= 0.001). In the period post-intervention there as a difference in the surface of RF (p= 0.001), in the antero-posterior oscillation of the LF (p= 0.01) and in the latero-lateral oscillation of the RF (p= 0.01). Regarding the gait velocity we observed before intervention a difference in the mean velocity of RF (p= 0.04). In the IG we observed a strong association before intervention between BDNF and GSH (r= 0,8; p=0,001), as well as between BDNF and physical activity (r= 0,7; p=0,03). We can conclude that controlled treadmill walking improves functional capacity of PD patients, accompanied by increased levels of antioxidant defenses.

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 141 - 176.

Perineural invasion (PNI) in cutaneous squamous cell carcinoma (cSCC) and desmoplastic melanoma (DM) may be a negative prognostic finding, and likely contributes to increased rates of local recurrence. The biological mechanisms underlying PNI remain unclear, although several lines of evidence implicate neurotrophins and their receptors. Expression of the high affinity nerve growth factor (NGF) receptor TrkA has been shown to be associated with PNI in numerous malignancies, although literature in cutaneous neoplasms is sparse. Given this, we sought to ascertain the incidence of PNI in a cohort cSCCs using double immunostaining (DIS), and to investigate PNI's relationship with TrkA expression and established histopathologic prognosticators. In DMs we investigated the relationship between TrkA and PNI. In DM we additionally analyzed expression of the low affinity NGF receptor (p75NGFR) and the presence of a functional polymorphism in the glial cell line-derived neurotrophic factor (GDNF) receptor RET (RETp) as they relate to PNI. In this IRB approved study, cSCCs from the head and neck (H&N) and 53 from non-H&N areas were immunohistochemically analyzed for PNI (DIS with S100 and p63) and TrkA expression. For DM, 43 cases were immunohistochemically evaluated for TrkA and p75NGFR expression while RETp was detected by direct DNA sequencing. The presence of each was correlated with histologically observed PNI. In cSCCs, comparing H&N versus non-H&N areas; using hemotoxylin and eosin (H&E) PNI was detected in 11% versus 6% of cases respectively and using DIS, in 23% versus 15% respectively, with significant disagreement between both methods (𝜅=0.47, p=0.002). There was a 2.33 fold increase in PNI detection with DIS compared to H&E (95%CI: 1.12-4.87; p=0.02). TrkA expression was 2.9 times more frequently observed in cSCCs from the H&N compared to those from non-H&N areas (p=0.01). Regardless of site, TrkA expression was associated with decreased degree of differentiation (OR=6.46, p=0.0006) and high-risk morphologic variants (OR = 6.53, p=0.002). TrkA expression was not significantly associated with PNI (p=0.33). In DM, PNI was present in 67% of cases. On univariate analysis; p75NGFR was associated with PNI (expression detected in 79% of PNI-positive cases compared to 36% of PNI-negative cases, p=0.005), increased Breslow's depth and greater Clark's Level (p= 0.007 and p= 0.01 respectively). RETp was noted in 28% of cases but was not significantly associated with PNI (p=0.27) or other histopathologic variables. TrkA expression was absent in all cases. PNI was associated with increased Breslow's depth and Clark's Level (p=0.01 and p=0.009 respectively). Controlling for the association between p75NGFR and depth, p75NGFR remained associated with an increased propensity for PNI (OR=4.68, p=0.04). In conclusion, increased PNI detection with DIS in cSCCs underscores the adjunctive utility of immunohistochemistry in microstaging. Although unlikely to play a role in the development of PNI, TrkA's association with cSCCs from H&N and select histopathologic parameters suggests a role for the NGF-TrKA axis in tumorogenesis while its absent expression in DM suggests that expression is lineage-related. Lastly, In DM, p75NGFR expression is significantly associated with PNI and a more locally aggressive phenotype.

No description available.

Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage cancer for the majority of patients. The work presented in this dissertation addresses the multifaceted role of glutamate in cancer cell-induced pain signalling and provides several potential therapeutic directions. Several cell types, including breast cancer cells and microglia, release glutamate via the system xC- antiporter. To limit the excitotoxic tendency of breast cancer cells to release glutamate in excess, we first indirectly inhibited xCT, the active subunit of system xC-, with the TrkA inhibitor AG879. We demonstrated that the system xC- antiporter is functionally influenced by the actions of nerve growth factor on its cognate receptor, TrkA, and that inhibiting this complex reduced CIP via downstream actions on xCT. Co-culture studies then demonstrated the direct effect of glutamate released by wildtype MDA-MB-231 carcinoma cells on microglial activation, as well as functional system xC- activity, while knockdown of xCT in MDA-MB-231 cells mitigated microglial activation and cystine uptake. Blockade of system xC- with sulfasalazine attenuated nociception in an immunocompetent murine model of CIP and inhibited tumour-induced microglial activation in the dorsal horn of the spinal cord. Finally, tumour-induced nociceptive behaviours appeared to progress in parallel with microglial activation in the hippocampus, and ablating microglia delayed the onset and severity of tumour-induced nociceptive behaviours, confirming that microglia are implicated in CIP and regional microglia are influenced by this pain. This is the first experimental evidence to demonstrate the effects of peripheral tumour on hippocampal microglial activation in relation to cancer-related nociception. These data collectively demonstrate that the system xC- antiporter is functionally implicated in CIP and may be particularly relevant to pain progression through spinal microglia. Upregulated xCT in chronically activated microglia may be one pathway to central glutamate cytotoxicity. Therefore, microglial xCT may therefore be a valuable target for mitigating CIP. / Thesis / Doctor of Philosophy (Medical Science) / Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage metastatic cancer. Clinically, achieving analgesia often comes at the expense of patients’ quality of life, as current therapeutics fail to adequately manage this pain and induce dose-dependent side effects. Cancer cells secrete excess amounts of glutamate, a signalling molecule involved in CIP, which can activate immune cells called microglia within the spinal cord. Mice that demonstrate tumour-induced pain exhibit an amplified immune response that manifests through the activation pattern and quantity of microglia within the spinal cord, as well as brain regions implicated in pain and distress. Pharmacologically blocking glutamate release from cancer cells limits this pain response, in addition to several physiological indicators of pain, including microglial activation in the central nervous system. Changes in microglia-related glutamate signalling may reflect the emotional problems reported by patients with CIP. Better understanding the mechanisms of CIP will help generate more comprehensive treatment approaches.

Emerging trends in cell-therapy based tissue repair have focused on the renewable source of adult stem cells including human bone marrow-derived mesenchymal stromal cells (hMSCs). Due to immunomodulatory properties as well as a potential to differentiate into cells characteristic of all three germ layers, hMSCs provide a source of immature cells for utilization in cell-therapy based treatments. Marrow isolated adult multilineage inducible (MIAMI) cells are a homogeneous sub-population of hMSCs which maintain self-renewal potential during ex vivo expansion, in addition to efficiently undergoing trans-differentiation into neuron-like cells in vitro. Even though hMSCs have the potential to be used for neural tissue repair, the molecular mechanisms by which they are stimulated to become neuron-like cells have not been fully characterized. Therefore the work described herein focuses on the molecular mechanisms by which MIAMI cells undergo NT-3 dependent neuronal commitment. MIAMI cells express both the full length (FL-) and tyrosine kinase deficient (TKd-) isoforms of the NTRK3 receptor, the primary NT-3 receptor, at the protein level. NT-3 stimulation of MIAMI cells during neuronal commitment induced the phosphorylation of FL-NTRK3, degradation of TKd-NTRK3, downstream activation of the Mek1/2-Erk1/2 signaling cascade, and subsequent up-regulation of a limited number of pro-neuronal genes. These findings were verified using chemical inhibitors to block NTRK autophosphorylation (K252a) and Erk1/2 activation (U0126). TKd-NTRK3 is hypothesized to activate Rac1 upon NT-3 stimulation. Rac1 was found to suppress NT-3 stimulated Erk1/2 phosphorylation, as well as downstream gene expression, as determined using a Rac1 chemical inhibitor. Further characterization confirmed that Rac1b is the predominant Rac1 isoform in MIAMI cells. Rac1b siRNA mediated knock-down resulted in increased expression of the pro-neuronal genes NGN2, MAP2, NFH and NFL during NT-3 stimulation via regulation of Mek1/2-Erk1/2. Rac1b is also involved in NT-3 stimulated cell proliferation, as well as repression of CCND1 and CCNB1 mRNA expression. In an attempt to enhance neuronal differentiation of MIAMI cells, EGF and bFGF were used to pretreat MIAMI cells prior to NT-3 stimulated neuronal commitment. EGF/bFGF pretreatment increased NTRK3 and NTRK1 protein levels along with NT-3 stimulated Erk1/2 phosphorylation. In addition, bFGF versus EGF/bFGF pretreatment restricted the expression of the pro-neuronal transcription factors Ngn2 and Prox1 versus the neural stem cells self-renewal transcription factor Musashi-1, respectively. The culmination of this work provides a model for the NT-3 induced neuronal commitment of MIAMI cells in vitro, as well as insight into the neurogenic potential of MSCs for future applications in cell-therapy based tissue repair.

<p>Understanding the mechanisms of limbic epileptogenesis in cellular and molecular terms may provide novel therapeutic targets for its prevention. The neurotrophin receptor tropomyosin-related kinase B (TrkB) is thought to be critical for limbic epileptogenesis. Enhanced activation of TrkB, revealed by immunodetection of enhanced phosphorylated TrkB (pTrkB), a surrogate measure of its activation, has been identified within the hippocampus in multiple animal models. Knowledge of the cellular locale of activated TrkB is necessary to elucidate its functional consequences. Using an antibody selective to pTrkB in conjunction with confocal microscopy and cellular markers, we determined the cellular and subcellular locale of enhanced pTrkB induced by status epilepticus (SE) evoked by infusion of kainic acid into the amygdala of adult mice. SE induced enhanced pTrkB immunoreactivity in two distinct populations of principal neurons within the hippocampus--the dentate granule cells and CA1 pyramidal cells. Enhanced immunoreactivity within granule cells was found within mossy fiber axons and giant synaptic boutons. By contrast, enhanced immunoreactivity was found within apical dendritic shafts and spines of CA1 pyramidal cells. A common feature of this enhanced pTrkB at these cellular locales is its localization to excitatory synapses between excitatory neurons, presynaptically in the granule cells and postsynaptically in CA1 pyramidal cells. Long-term potentiation (LTP) is one cellular consequence of TrkB activation at these excitatory synapses that may promote epileptogenesis.</p><p>The importance of TrkB in diverse neuronal processes, as well as its involvement in various disorders of the nervous system, underscores the importance of understanding how it is activated. The canonical neurotrophin ligand which activates TrkB is brain derived neurotrophic factor (BDNF). Zinc, however, has also been demonstrated to activate this receptor through a mechanism whereby it does not directly interact with it, known as transactivation. Presynaptic vesicles of mossy fiber boutons of stratum lucidum are particularly enriched in zinc, where it is co-released with glutamate in an activity dependent fashion, and incorporated into these vesicles by the zinc transporter, ZnT3. Given the presence of large quantities of zinc within stratum lucidum, we hypothesized that this metal may contribute to TrkB transactivation at this locale. To this end, we examined the contributions of both BDNF and synaptic vesicular zinc to TrkB activation in stratum lucidum of mouse hippocampus under physiological conditions. Utilization of mice which are genetic knockouts for BDNF and/or ZnT3 allowed us to examine TrkB activation in the absence of one or both of these ligands. This was done using an antibody for pTrkB in conjunction with confocal microscopy, assaying immunoreactivity at the cellular and synaptic locales within stratum lucidum where pTrkB was previously found to be enriched. Our results suggest that BDNF contributes to TrkB activation within stratum lucidum. Interestingly, ZnT3 mice displayed an increase in BDNF protein and TrkB activation, demonstrating that synaptic zinc regulates BDNF and TrkB signaling at this locale.</p> / Dissertation

No / Nerve growth factor (NGF) has long been recognized as an important mast cell (MC) growth factor. To explore whether other neurotrophins (NTs) of the NGF family, which are widely expressed in mouse skin, affect the numbers and/or functions of MCs we examined the effects of NT-3 on neonatal skin MCs. We demonstrate that TrkC, the high affinity NT-3 receptor, is expressed by virtually all neonatal skin MCs in C57BL/6 mice, which indicates that MCs can respond to NT-3. Skin of neonatal and early postnatal NT-3-overexpressing mice (promoter: K14) displayed significantly and up to twofold increased numbers of MCs during the first 20 days after birth, as compared to wild-type mice. To check whether this increase in MC numbers in NT-3 transgenic mice reflects a higher rate of proliferation, we performed immunohistochemistry, which revealed that only 1-2% of all skin MCs both in NT-3-overexpressing and in wild-type controls showed Ki-67-positive nuclei, suggesting that the observed differences in the number of MCs do not reflect a higher rate of MC proliferation. Additionally, we show that the effect of NT-3 on the number of MCs is most likely to be stem cell factor (SCF)-independent, because NT-3 significantly downregulates secretion of SCF-protein in cultured dermal fibroblasts, as assessed by enzyme-linked immunosorbent assay. Numbers of skin MCs in neonatal TrkC-deficient mice were found to be modestly reduced, as compared to wild-type mice, indicating that NT-3 can modulate the number of MCs directly via TrkC, although TrkC does not seem to be essential for the number of basal MCs. To further analyze the effects of NT-3 on MCs, we stimulated skin organ culture of early postnatal C57BL/6 mouse skin with 5-50 ng/ml NT-3, which induced a significant increase in MC degranulation, as visualized by Giemsa staining. However, stimulation of isolated neonatal dermal skin MCs with NT-3 in vitro failed to result in MC activation, as measured by serotonin release. Our data suggest a role for NT-3 in the maturation of MCs, such as a TrkC-mediated stimulation of the differentiation of pre-existing, less mature MCs and/or by enhancing the migration of circulating MC precursors into the skin.

Aside from preventing traumatic brain injuries (TBIs) altogether, treatment options for TBI typically focus on the secondary biochemical processes that occur in response to the primary mechanical insult. These secondary injuries can lead to apoptosis and necrosis in the days and weeks that follow a TBI. Therefore, finding a treatment that can prevent, reduce, and repair secondary damage is instrumental in the recovery of TBI patients. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) has been identified as a TrkB agonist that mimics the effects of brain derived neurotrophin factor (BDNF). Upon binding to the TrkB receptor, signaling cascades are initiated that can promote neuronal survival and neural differentiation. The use of 7,8-DHF in the treatment of TBI is favorable due to its long half-life and ability to pass the blood-brain barrier (BBB). In this study, we evaluated the dosage time frame of 7,8-DHF that would allow for the greatest impact in recovery after a focal TBI. Adult Sprague-Dawley rats were subjected to a moderate cortical impact injury and administered a 5mg/kg dose of 7,8-DHF i.p. for five days starting on day 0, 2, 3, or 5 post injury. Sensorimotor function was evaluated with beam walk and rotarod test. Morris Water Maze (MWM) and fear conditioning test were used to analyze cognitive function. Biotinylated dextran amine (BDA) was injected into the contralateral cerebral cortex 14 days after injury and animals were sacrificed 28 dpi. Brain sections were processed for Giemsa histological staining to assess cortical lesion volume and the total number of surviving neurons. Parallel sections were processed for BDA staining to assess changes of axon sprouting in the injured cortex. VGlut-1 staining of the hippocampus was used to identify presynaptic plasticity. We found that the administration of 7,8- DHF starting at one hour after TBI could provide protection against motor and cognitive dysfunction. Histological examination showed a significant reduction of cortical lesion volume and higher number of survival neurons in the injured hippocampus when 7,8-DHF administration began one hour and two days after injury. BDA staining of intracortical axon sprouting and VGlut-1 staining of the hippocampus highlighted a trend that 7,8-DHF administration starting day five post brain injury may enhance neuronal plasticity. Collectively, the results indicate that 7,8-DHF can provide the better neuronal protection when administration begins one hour after TBI.