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Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 97 pages. Includes Vita. Includes bibliographical references.

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly population and is believed to be caused by the overproduction and accumulation of the toxic amyloid beta (Aβ) peptide in the brain. Aβ is produced by two separate enzymatic cleavage events of the larger membrane bound amyloid precursor protein, APP. The first, and rate-limiting, cleavage event is made by beta-secretase, or BACE1, and is thus an attractive therapeutic target. Our lab, as well as many others, has shown that BACE1 protein and activity are increased in late-stage sporadic AD. We have extended these findings to show that BACE1 is increased in the earliest stages of AD before the onset of significant Aβ accumulation, indicating a potential causal role in the disease. Interestingly, BACE1 mRNA levels are unchanged in AD, leading to reason that a post-transcriptional method of BACE1 regulation is altered in disease. To date, the mechanism for this aberrant post-transcriptional regulation has not been elucidated. This study has implicated the cellular nucleic acid binding protein (CNBP), a highly conserved RNA binding protein, as a positive regulator of BACE1 translation, with implications for the etiology of sporadic AD. CNBP overexpression in cultured cells or spiked into a cell-free in vitro translation system increased BACE1 protein expression without affecting BACE1 mRNA levels. Knockdown of CNBP reduced BACE1 protein and mRNA slightly. Furthermore, CNBP associated with BACE1 mRNA in cell lysates and bound directly to the BACE1 5’ UTR in vitro, which confers most of the regulatory activity. Importantly, CNBP was increased in the progression of AD and correlated with BACE1 expression. Cellular stressors (such as glucose deprivation and oxidative stress) that occur in the AD brain increase BACE1 translation and we have found that these stressors increased CNBP expression as well. Early experimental evidence suggests that CNBP may enhance BACE1 translation through a cap-independent mechanism, which is an alternative translational pathway activated by cell stress. These studies indicate that the RNA binding protein CNBP is a novel trans-acting factor important for the regulation of BACE1 protein production and may be a viable therapeutic target for AD.

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic cell death in the substantia nigra pars compacta (SNc) and dopamine (DA) depletion in the striatum. Current pharmacological treatments are aimed at the replacement of striatal DA via the administration of levodopa. While this therapy is beneficial initially, long-term treatment is associated with significant side effects, and disease progression continues. The present experiments investigate neuroprotective and neurotransplantation strategies as alternatives to palliative pharmacologic treatments. The optimal therapeutic approach to neurodegenerative diseases would be to protect against cell death and prevent disease progression. PD is well-suited for such neuroprotective strategies as primarily one cell population is affected in this disorder. Neurotrophic factors (NTFs) have been identified which support dopaminergic neuronal survival in vitro. In the present studies, the neuroprotective effects of the neurotrophin brain-derived neurotrophic factor (BDNF) have been evaluated in a 1-methyl-4-phenylpyridinium (MPP+) model of substantia nigra (SN) degeneration. BDNF-secreting fibroblasts were implanted dorsal to the SN prior to the infusion of the mitochondrial complex I inhibitor MPP+. Subsequent histological analysis demonstrated that BDNF is able to attenuate MPP+ induced dopaminergic cell loss in the SNc. Moreover, neurochemical evaluation demonstrated that BDNF is able to enhance DA levels in the remaining SN neurons in this same paradigm. The cause of cell death in neurodegenerative diseases likely involves the interaction of mitochondrial impairment, excitotoxicity, and oxidative stress. In order to evaluate the mechanism of NTF-mediated protection, the ability of nerve growth factor (NGF) to attenuate the production of the oxidant peroxynitrite was evaluated in a model of mitochondrial impairment. NGF was found to decrease the production of 3-nitrotyrosine, the product of peroxynitrite mediated tyrosine nitration. Thus, NTF-mediated neuroprotection may act in part by decreasing reactive oxygen species and oxidative stress. At present, neuroprotective therapies are not clinically available. An alternate therapeutic approach to PD is the replacement of striatal DA and reconstruction of synaptic circuitry via the intrastriatal transplantation of fetal dopaminergic neurons. Current transplantation protocols using human fetal tissue are constrained by limited tissue availability. In order to investigate an alternate cell source for the treatment of PD, fetal porcine dopaminergic neurons were implanted into the DA depleted striatum of 6-OHDA lesioned rats. Amphetamine-induced rotational recovery was monitored, and graft survival was evaluated 19 weeks after grafting. In immunosuppressed rats, porcine dopaminergic neurons were found to attenuate rotational deficits and extensively reinnervate the host striatum. The neuroprotective effects of BDNF suggest that NTFs may be important mediators of dopaminergic neuronal survival and function in the adult brain. However, several conditions including appropriate dosage and delivery need to be determined before clinical applications may be achieved. As an alternative to neuroprotection, neurotransplantation not only restores striatal DA but also reconstructs the synaptic circuitry of the basal ganglia. The finding that porcine dopaminergic neurons survive with in adult host brain, reinnervate the DA depleted striatum, and mediate functional recovery suggests that porcine DA neurons may serve as an alternate cell source for transplantation in PD.

Spontaneous mutations in the human LIS1 gene are responsible for Type I lissencephaly ("smooth brain"). The distribution of neurons within the cerebral cortex of lissencephalic children appears randomized, probably owing to a defect in neuronal migration during early development. LIS1 has been implicated in the dynein pathway by genetic analyses in fungi. We previously reported that the vertebrate LIS1 co-localized with dynein at prometaphase kinetochores, and interference with LIS1 function at kinetochore caused misalignment of chromosomes onto the metaphase plate. This leads to a hypothesis that LIS1 might regulate kinetochore protein targeting. In order to test this hypothesis, I created dominant inhibitory constructs of LIS1. After removal of the endogenous LIS1 from the kinetochore by overexpression of the N-terminal self-association domain of LIS1, dynein and dynactin remained at the kinetochores. This result indicated that LIS1 is not required for dynein to localize at the kinetochore. Next, CLIP-170 was displaced from the kinetochores in the LIS1 full-length and the C-terminal WD-repeat overexpressers, suggesting a role for LIS1 in targeting CLIP-170 onto kinetochores. LIS1 was co-immunoprecipitated with dynein and dynactin. Its association with kinetochores was mediated by dynein and dynactin, suggesting LIS1 might interact directly with subunits of dynein and/or dynactin complexes. I found that LIS1 interacted with the heavy and intermediate chains (HC and IC) of dynein complex, and the dynamitin subunit of dynactin complex. In addition to kinetochore targeting, the LIS1 C-terminal WD-repeat domain was responsible for interactions with dynein and dynactin. Interestingly, LIS 1 interacted with two distinct sites on HC: one in the stem region containing the subunit-binding domain, and the other in the first AAA motif of the motor domain, which is indispensable for the ATPase function of the motor protein. This LIS1-dynein motor domain interaction suggests a role for LIS1 in regulating dynein motor activity. To test this hypothesis, changes of dynein ATPase activity was measured in the presence of LIS1 protein. The ATPase activity of dynein was stimulated by the addition of a recombinant LIS1 protein. Besides kinetochores, others and we have found LIS1 also localized at microtubule plus ends. LIS1 may mediate dynein and dynactin mitotic functions at these ends by interacting with astral microtubules at cortex, and associating with the spindle microtubules at kinetochores. Overexpression of LIS1 displaced dynein and dynactin from the microtubule plus ends, and mitotic progression was severely perturbed in LIS1 overexpressers. These results suggested that the role for LIS1 at microtubule plus ends is to regulate dynein and dynactin interactions with various subcellular structures. Results from my thesis research clearly favored the conclusion that LIS1 activates dynein ATPase activity through its interaction with the motor domain, and this activation is important to establish an interaction between dynein and microtubule plus ends during mitosis. I believe that my thesis work not only has provided ample implications regarding dynein dysfunction in disease formation, but also has laid a significant groundwork for more future studies in regulations of the increasing array of dynein functions.

One of the consequences of traumatic brain injury is the postconcussion syndrome. The symptoms in this syndrome include headache, dizziness, poor memory, poor concentration, easy fatigue, drowsiness, irritability, sensitivity to light, sensitivity to noise, low alcohol tolerance, visual problems, auditory problems, nausea, vomiting, anxiety, and depression. Several factor analytic studies have shown that these symptoms load onto cognitive and noncognitive factors (Bohnen, Twijnstra, & Jolles, 1992). The aim of this study was to determine whether patients who report different symptoms also evidence differences in cognitive deficits, as indexed by reaction time. For this purpose 106 subjects (mean age 25.92 years; SD=6.05) of both sexes were tested on 8 reaction time tasks adapted from Shum, McFarland, Bain, and Humphreys (1990). There were 54 traumatic brain injury patients (mean age 26.40 years; SD=6.23) drawn from three Pretoria hospitals. They were heterogeneous with respect to diagnosis and severity of injury. For the controls (N=52), the mean age was 25.43 years (SD=5.88). The eight reaction time tasks constituted 4 task variables, each with 21evels. From these tasks, 36 reaction time indexes were derived. The indexes were classified into 4 groups, viz., reaction time (RT), movement time (MT), total reaction time (TT), and subtraction scores (SB, the difference between the 2 levels for each task variable). RT reflects the decision component and MT reflects the response execution component of reaction time. Partial correlation coefficients for all symptoms (p0,01) showed that some symptoms were most frequently associated with RT whilst others were most frequently associated with MT. On factor analysis with varimax rotation, symptoms loaded predominantly with SB scores. Symptoms also loaded with different task variablseuiggesting that they correlated with deficits on different stages of information processing. Taking into account possible methodological constraints that were discussed, these results confirm that different symptoms within the postconcussion syndrome correlate with different cognitive deficits. The correlations between symptoms and indices of reaction time are moderated by the characteristics of the symptoms (frequency & intensity), and the duration since injury. These findings have significance for understanding the aetiology of the postconcussion symptoms and for planning treatment. / Psychology / Ph. D. (Psychology)

Made available in DSpace on 2015-06-17T19:34:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-04. Added 1 bitstream(s) on 2015-06-18T12:46:48Z : No. of bitstreams: 1 000831507.pdf: 1599546 bytes, checksum: 662ec2cc8a79f99b1f64c6a60a89c9ff (MD5) / A neurorrafia látero-terminal sem lesão do nervo doador representou grande avanço na área de microcirurgia de nervos periféricos. O coto distal do nervo receptor é suturado na face lateral do nervo doador, sem prejudicá-lo. Desde 1994, o FK-506, uma droga imunossupressora, tem sido utilizado para impedir a rejeição do aloenxerto nos transplantes de órgãos e apresentou, em alguns estudos, como ação secundária, efeitos positivos sobre a regeneração nervosa periférica. O objetivo desse trabalho foi estudar se a administração de FK-506 traria benefícios na regeneração do nervo após neurorrafia látero-terminal. Foram utilizados 80 ratos Wistar, divididos em 4 grupos experimentais. Cada rato teve seu nervo fibular esquerdo seccionado e a extremidade distal suturada término-lateralmente ao nervo tibial. Os animais do grupo I, II e III foram submetidos à administração de FK-506 com dose de 1,0, 0,5 e 0,25 mg/kg/dia, respectivamente, durante dois meses, enquanto os animas do grupo Controle (GC) não receberam nenhuma droga. Após dois meses da cirurgia, os ratos foram submetidos ao teste da marcha, estudo eletrofisiológico e análise das fibras musculares e nervosas. O grupo II (GII) apresentou o músculo tibial cranial (MTC) com menor massa (p < 0,05) e maior amplitude (p = 0,019). Os resultados do número total de fibras nervosas foram maiores para GII (p < 0,001). Não houve diferença significativa entre os grupos para os índices funcionais. Com base em nosso modelo experimental, pudemos concluir que a administração de FK-506 na dose de 0,5 mg/Kg/dia diminuiu o ganho de massa corporal e massa do músculo tibial cranial e aumentou a regeneração das fibras nervosas, embora não tenha conseguido alterar a resposta funcional / The latero-terminal neurorrhaphy without injury to the donor nerve, represented great advances in microsurgery of peripheral nerves. The receptor nerve distal stump was sutured on the lateral side of the donor nerve, without harming it. Since 1994, FK-506, an immunosupressive drug, has been used to prevent allograft rejection in organ transplants and presented, in some studies, as a secondary action, positive effects on peripheral nerve regeneration. The aim of this work was to investigate whether the administration of FK-506 would benefit nerve regeneration after a latero-terminal neurorrhaphy. We used 80 Wistar rats, divided into four experimental groups. Each rat had its left fibular nerve sectioned and the distal stump sutured to the lateral of the tibial nerve. The animals of group I, II and III were subjected to the administration of FK-506 in an amount of 1.0, 0.5 to 0.25mg/K/day, respectively, for two months, while the aminals of the control group received no drug. Two months after surgery the rats were submitted to the walking test, eletrophysiological study and analysis of muscle and nerve fibers. The group II (GII) showed cranial tibial muscle (MTC) with lower mass (p < 0.05) and higher amplitude (p = 0.019). The results by the total number of nerve fibers was higher for GII (p < 0.001) . There was no significant difference between groups for functional indices. Based on our experimental model, we could conclude that the administration of 0.5 mg/Kg/dia of FK-506 decreased body mass gain and mass of MTC and increased regeneration of nerve fibers, although not able to change the functional response

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