<p>Bed bugs (<i>Cimex lectularius</i> L.) are globally important human ectoparasites. Their management necessitates the use of multiple control techniques. Plant-derived essential oils are extracts from aromatic plants that represent one of the alternative control measures for bed bug control, in addition to mechanical options and synthetic pesticides. However, there is limited information available on the efficacy and toxicology of plant essential oils against bed bugs. This project was designed with the aim to provide in-depth information on efficacy, toxicology and mode-of-action of essential oils and their insecticidal constituents in bed bugs. Initially, I evaluated topical and fumigant toxicity of fifteen essential oil components against adult male bed bugs of the Harlan strain (an insecticide susceptible strain). Neurological effects of the six most toxicologically active compounds were also determined. In both topical and fumigant bioassays, carvacrol and thymol were the most active compounds. Spontaneous electrical activity measurements of the bed bug nervous system demonstrated neuroinhibitory effects of carvacrol, thymol and eugenol, whereas linalool and bifenthrin (a pyrethroid class insecticide) produced excitatory effects. Further, I evaluated the efficacy and neurological impacts of a mixture of three neuroinhibitory compounds; carvacrol, eugenol and thymol in 1:1:1 ratio against adult male bed bugs of the Harlan strain. This mixture of monoterpenoids as well as the mixture of synthetic insecticides exhibited a synergistic affect in topical bioassays. In electrophysiology experiments, the monoterpenoid mixture led to higher neuroinhibitory effects, whereas a mixture of synthetic insecticides caused higher neuroexcitatory effects in comparison to single compounds. </p> <p>In the next objective of my dissertation, I compared the efficacy of five plant essential oils (thyme, oregano, clove, geranium and coriander), their major components (thymol, carvacrol, eugenol, geraniol and linalool) and EcoRaider^® (commercial product) between pyrethroid susceptible (Harlan) and field collected (Knoxville) bed bug populations. Initially, I found that the Knoxville strain was 72,893 and 291,626 fold resistant to topically applied deltamethrin (a pyrethroid class insecticide) compared to the susceptible Harlan strain at the LD₂₅ and LD₅₀ lethal dose levels, respectively. Synergist bioassays and detoxification enzyme assays showed that the Knoxville strain possesses significantly higher activity of cytochrome P450 and esterase enzymes. Further, Sanger sequencing revealed the presence of the L925I mutation in the voltage gated sodium channel gene. The Knoxville strain, however, did not show any resistance to plant essential oils, their major components or EcoRaider^® in topical bioassays (resistance ratios of ~ 1). In the final objective, I evaluated the efficacy of binary mixtures of above-mentioned essential oils or their major components or EcoRaider^® with deltamethrin in susceptible and resistant bed bugs. In topical application bioassays, binary mixtures of essential oils or their major components or EcoRaider^® and deltamethrin at the LD₂₅ dose caused a synergistic increase in toxicity in resistant bed bugs. Further, I studied the inhibitory effects of major essential oil components on detoxification enzyme activities (cytochrome P450s, esterases and glutathione transferases). Detoxification enzyme assays conducted using protein extracts from bed bugs pre-treated with essential oil constituents showed that these compounds significantly inhibited cytochrome P450 activity in the resistant strain, but esterase and glutathione transferase activity were unaffected. No inhibition of detoxification enzyme activities was observed in the Harlan strain bed bugs pre-treated with essential oil constituents.</p> <p>In conclusion, my dissertation research has created the foundation for utilization of natural products for bed bug management by (i) describing the efficacy of plant essential oils and their components against bed bugs, (ii) discovering synergistic interactions between essential oil components at the nervous system level, (iii) determining susceptibility of deltamethrin-resistant bed bugs to plant essential oils and their constituents and (iv) identifying synergistic effects of essential oils or their components on toxicity of pyrethroid insecticides and underlying mechanisms of this synergistic interaction. </p> <br>

Indiana University-Purdue University Indianapolis (IUPUI) / LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from cognitive impairment to craniofacial abnormalities. While DS originates from the trisomy of human chromosome 21 and FAS from prenatal alcohol consumption, many of the defining characteristics for these two disorders are stunningly similar. A survey of the literature revealed over 20 similar craniofacial and structural deficits in both human and mouse models of DS and FAS. We hypothesized that the similar phenotypes observed are caused by disruptions in common molecular or cellular pathways during development. To test our hypothesis, we examined morphometric, genetic, and cellular phenotypes during development of our DS and FAS mouse models at embryonic days 9.5-10.5. Our preliminary evidence indicates that during early development, dysregulation of Dyrk1a and Rcan1, cardinal genes affecting craniofacial and neurological precursors of DS, are also dysregulated in embryonic FAS models. Furthermore, Caspase 3 was also found to have similar expression in DS and FAS craniofacial neural crest derived tissues such as the first branchial arch (BA1) and regions of the brain. This may explain a developmental deficit by means of apoptosis. We have also investigated the expression of pAkt, a protein shown to be affected in FAS models, in cells located within the craniofacial precursor of Ts65Dn. Recent research shows that Ttc3, a gene that is triplicated and shown to be overexpressed in the BA1 and neural tube of Ts65Dn, targets pAkt in the nucleus affecting important transcription factors regulating cell cycle and cell survival. While Akt has been shown to play a role in neuronal development, we hypothesize that it also affects similar cellular properties in craniofacial precursors during development. By comparing common genotypes and phenotypes of DS and FAS we may provide common mechanisms to target for potential treatments of both disorders. One of the least understood phenotypes of DS is their deficient immune system. Many individuals with DS have varying serious illnesses ranging from coeliac disease to respiratory infections that are a direct result of this immunodeficiency. Proteasomes are an integral part of a competent and efficient immune system. It has been observed that mice lacking immunoproteasomes present deficiencies in providing MHC class I peptides, proteins essential in identifying infections. A gene, Psmg1 (Dscr2), triplicated in both humans and in Ts65Dn mice, is known to act as a proteasome assembly chaperone for the 20S proteasome. We hypothesized that a dysregulation in this gene promotes a proteasome assembly aberration, impacting the efficiency of the DS immune system. To test this hypothesis we performed western blot analysis on specific precursor and processed β-subunits of the 20S proteasome in thymic tissue of adult Ts65Dn. While the β-subunits tested displayed no significant differences between trisomic and euploid mice we have provided further insight to the origins of immunodeficiency in DS.

Spinal neurons are important in several aspects motor control. For example, the neurons essential for locomotor movements reside in the ventral spinal cord. In this thesis, different motor control functions are being related to neuronal populations defined by their common expression of a gene. First, a targeted disruption of the gene for vesicular glutamate transporter 2 (Vglut2/ Slc17a6) is described. The mutant animals die at birth because of their inability to breathe. The neuronal network in the brainstem, responsible for inspiration, was shown to become non-functional by the targeted deletion of Vglut2. To our surprise, it was still possible to induce rhythmic activity with normal left/right alternation in spinal cords isolated from VGLUT2-null embryos. Inconsistent reports of Vglut1 expression in the spinal cord made us re-evaluate the Vglut1 and Vglut2 expressions. While Vglut2 expression was widespread in the spinal cord, Vglut1 expression was restricted to a few cells dorsal to the central canal.  Taken together, the data suggest that, glutamatergic signaling is mandatory to drive the bilateral breathing, but not needed for coordination of basal alternating spinal locomotor rhythm. Next, a screen for genes with restricted ventral expression was made. Some of the genes found could be connected to the characteristics of specific neuronal cell populations. For example, fast motor neurons were shown to express the genes Calca and Chodl. Further, we found the Chrna2 expression selectively in putative Renshaw cells. It seems likely that the gene product, the alpha2 subunit of the nicotinergic receptor, could be linked to the unique connection of motor neurons to Renshaw cells. We used the Chrna2 promoter to drive expression of Cre recombinase in a transgenic mouse. The Cre activity was present in most neurons labeled with Renshaw cell markers, which should make it a useful tool for functional studies of this population. The studies presented here show how the genes expressed in subsets of neurons can be used to target populations of neurons for functional studies of neuronal systems.

Questions : Cette thèse visait à répondre à deux questions fondamentales : 1) Est-ce que les athlètes qui présentent un historique de commotions cérébrales du sport en conservent des effets délétères à long terme? ; et 2) Est-ce que les effets néfastes des commotions cérébrales récurrentes sur le fonctionnement tant cognitif que moteur sont cumulatifs? Devis expérimental : À l’aide d’un plan d’investigation double-cohorte réalisé avec un groupe d’athlètes évoluant au niveau universitaire et un autre formé d’anciens athlètes universitaires testés plus de trois décennies plus tard, les quatre études qui composent cette thèse ont employé des méthodes raffinées d’investigation des fonctions cognitives et motrices pour en déceler des atteintes persistantes. Méthodologie : Les potentiels évoqués cognitifs ainsi que les tests neuropsychologiques ont permis de sonder le fonctionnement cognitif de ces athlètes alors que la stimulation magnétique transcrânienne, une plateforme de force permettant de mesurer la stabilité posturale ainsi qu’un système d’enregistrement tridimensionnel des mouvements rapides alternatifs ont servi à l’évaluation de l’intégrité du système moteur. Résultats : Cette thèse a permis de déceler des altérations persistentes et cumulatives des fonctions cognitives et motrices. De plus, ces subtiles atteintes observées chez les jeunes athlètes, affectant essentiellement des marqueurs neurophysiologiques sous-cliniques du fonctionnement cognitif et moteur, s’étaient accentuées chez les anciens athlètes universitaires qui montraient un déclin quantifiable tant des fonctions cognitives que motrices. Discussion : Ces résultats suggèrent d’une part que les commotions cérébrales du sport entraînent des altérations cognitives et motrices chroniques qui s’accentuent en fonction du nombre de commotions cérébrales subies. D’autre part, les effets délétères des commotions cérébrales du sport sur le fonctionnement cognitif et moteur combinés à ceux associés au processus de vieillissement entraînent un déclin cognitif et moteur quantifiable en comparaison aux anciens athlètes n’ayant jamais subi de commotions cérébrales. / Question: This thesis aimed to address two fundamental issues: 1) Are there long-lasting effects of sports-related concussion on cognitive and motor functions? and 2) Are the adverse effects of recurrent concussions cumulative? Experimental Design: The cross-sectional thesis design included a group of active university-level athletes as well as a group of former athletes recruited more than three decades after their university years who were tested on neurophysiological measures of both cognitive and motor system functions. Methods: Event-Related potentials and neuropsychological tests were used to assess cognitive functions while transcranial magnetic paradigms were used to assess motor cortex excitability, a force platform was used to assess postural stability and a 3-dimensional recording device was used to track hand position when performing a rapid alternating movement task. Results: This thesis disclosed persistent and cumulative alterations of both cognitive and motor functions after sports concussions. Furthermore, subclinical, neurophysiological alterations found in young concussed athletes were exacerbated in former athletes with concussions who displayed quantifiable cognitive and motor functions decline more than three decades post-concussion. Discussion: These results suggest that sports concussions induce cognitive and motor functions abnormalities that worsen as a function of the number of concussions sustained. Moreover, findings from the present thesis indicate that the deleterious effects of sports concussion on cognitive and motor system functions combined to those associated with the aging process lead to quantifiable decline on both cognition and motor functions.

The influence of experience during brain development was investigated on juvenile behavior, adult amphetamine sensitization, and neuronal structural plasticity in rats. Two experiential factors (i.e., tactile stimulation and stress) were studied either before or soon after birth. Early experience feminized social behavior in males; however, only stress enhanced anxiety-like behavior in males. Repeated amphetamine administration resulted in the development and persistence of behavioral sensitization. However, tactile stimulation attenuated the drug-induced behavioral sensitization whereas stress failed to influence the degree of sensitization. Neuroanatomical findings revealed that early experience altered the cortical and subcortical structures. Furthermore, drug exposure reorganized the brain structures involved in addiction but early experience prevented the drug-associated changes. Early adverse experience influences the subsequent exposure to a drug of abuse at anatomical level whereas a favorable experience has an effect both at behavioral and anatomical levels and thus may play a protective role against drug-induced sensitization and addiction. / xii, 263 leaves : ill. ; 29 cm

Synaptic plasticity describes efficacy changes in synaptic transmission and ranges in duration from tens to hundreds of milliseconds (short-term), to hours and days (long-term). Short-term plasticity plays crucial roles in synaptic computation, information processing, learning, working and short-term memory as well as its dysfunction in psychiatric and neurodegenerative diseases. The main aim of my PhD thesis was to determine the molecular mechanisms of different forms of presynaptic plasticity. Short-term facilitation increases neurotransmitter release in response to a high-frequency pair (paired-pulse facilitation; PPF) or train (train facilitation; TF) of presynaptic stimuli. Synaptotagmin 7 (Syt7) has been shown to act as residual calcium (Ca$_{res}$) sensor for PPF and TF at various synapses. Syt7 also seems to be involved in recovery from depression, whereas its role in neurotransmission remains controversial. My aim was to express Syt7 in a synapse where it is not normally found and determine how it affects short-term synaptic plasticity. Immunohistochemistry indicated that Syt7 is not localized to cerebellar climbing fibers (CFs). Wild-type (WT) and Syt7 knockout (KO) recordings at CF to Purkinje cell (CF-PC) synapses established that at near-physiological external calcium (Ca$_{ext}$) levels both genotypes displayed similar recovery from paired-pulse depression. In low Ca$_{ext}$,WT CF-PC synapses showed robust PPF, which turned out to be independent of Syt7. All my experiments strongly suggested that WT CFs do not express native Syt7, but display low Ca$_{ext}$ CF-PC PPF and TF. Thus, channelrhodopsin-2 and Syt7 were bicistronically expressed via AAV9 virus in CFs. This ectopic Syt7 expression in CFs led to big increases in low-Ca$_{ext}$ CF-PC facilitation, more than doubling PPF and more than tripling TF. While overexpression of Syt7 might turn out to have an effect on the initial release probability (pr), the observed CF-PC facilitation increase still critically depended on presynaptic Syt7 expression. And when comparing only cells in a defined EPSC1 amplitude range, the Syt7-induced increase in low-Ca$_{ext}$ PPF could not be accounted for by changes in initial pr, suggesting a general role for Syt7 as calcium sensor for facilitation. Another form of short-term plasticity, post-tetanic potentiation (PTP), is believed to be mediated presynaptically by calcium-dependent protein kinase C (PKC) isoforms that phosphorylate Munc18-1 proteins. It is unknown how generally applicable this mechanism is throughout the brain and if other proteins might be able to modulate PTP. Combining genetic (PKCαβy triple knockout [TKO] and Munc18-1SA knock-in [Munc18 KI] mice, in which Munc18- 1 cannot get phosphorylated) with pharmacological tools (PKC inhibitor GF109203), helped us show that PTP at the cerebellar parallel fiber to Purkinje cell (PF-PC) synapse seems to depend on PKCs but seems mostly independent of Munc18-1 phosphorylation. In addition, compared to WT animals, genetic elimination of presynaptic active zone protein Liprin-α3 led to similar PF-PC PTP and paired-pulse ratios (PPRs). At the hippocampal CA3-CA1 synapse previous pharmacological studies suggested that PKC mediates PTP. A genetic approach helped to show that calcium dependent PKCs do not seem to be required for CA3-CA1 PTP. Pharmacologically inhibiting protein kinase A as well as genetically eliminating Syt7 also had no effect on CA3-CA1 PTP. In addition, Ca IM-AA mutant mice, in which Ca$_{v}$2.1 channels have a mutated IQ-like motif (IM) so that it cannot get bound by calcium sensor proteins any more, not only displayed regular PTP, but also normal PPF and TF at CA3-CA1 synapses. In conclusion, my PhD thesis helped further characterize different forms of presynaptic plasticity, underlined that short-term synaptic plasticity can be achieved through diverse mechanisms across the Mammalian brain and supported a potentially general role for synaptotagmin 7 acting as residual calcium sensor for facilitation.

Submitted by Emanuel Varela Cardoso (emanuel.varela@ufcg.edu.br) on 2018-06-29T20:52:05Z No. of bitstreams: 1 JOSÉ ALBERTO CAMPOS DA SILVA JÚNIOR – DISSERTAÇÃO (UAEMa) 2015.pdf: 3855504 bytes, checksum: 02bbfa20b694c9416d048edd8410bfd4 (MD5) / Made available in DSpace on 2018-06-29T20:52:05Z (GMT). No. of bitstreams: 1 JOSÉ ALBERTO CAMPOS DA SILVA JÚNIOR – DISSERTAÇÃO (UAEMa) 2015.pdf: 3855504 bytes, checksum: 02bbfa20b694c9416d048edd8410bfd4 (MD5) Previous issue date: 2015-03-17 / O monitoramento neurofisiológico intraoperatório (MNIO) é uma metodologia que agrega diferentes testes neurofisiológicos para uso simultâneo ou alternado num mesmo paciente durante o procedimento cirúrgico, podendo avaliar a neurofisiologia clínica em três campos: eletroencefalografia (EEG), eletromiografia (EMG) e potenciais evocados (PE). O potencial evocado visual (PEV) permite avaliar a função e integridade das estruturas corticais e subcorticais da via visual. Este exame é realizado cotidianamente nos laboratórios de neurofisiologia, auxiliando o esclarecimento de diferentes tipos de acometimentos da visão, seja por doença ou traumatismos. Este trabalho tem como objetivo desenvolver um protótipo para o monitoramento do PEV para avaliação da integridade das vias visuais durante cirurgias neurofisiológicas. Foram realizadas várias etapas para o desenvolvimento do protótipo do dispositivo ocular: simulação computacional; estudo das características do olho humano; prototipagem rápida; caracterização do Biopolímero ácido polilático (PLA) utilizado na prototipagem; desenvolvimento de um protótipo de dispositivo ocular com sistema de iluminação integrado por LEDs a realização dos testes de PEV com o protótipo desenvolvido. O PLA utilizado na prototipagem para o desenvolvimento de partes do dispositivo ocular foi caracterizado por Difração de Raios X (DRX), Espectroscopia na Região do Infravermelho por Transformada de Fourier (FTIR), Calorimetria Exploratória Diferencial (DSC), Microscopia Ótica (MO), Microscopia Eletrônica de Varredura (MEV) e Espectroscopia de Energia Dispersiva (EDS), esses resultados mostraram que a prototipagem rápida não alterou as propriedades físico-químicas e morfológicas do PLA. A simulação computacional forneceu parâmetros adequados ao desenvolvimento do dispositivo ocular que possibilitou uma maior eficiência na montagem do circuito eletrônico. Os resultados dos testes de PEV foram realizados em diferentes pacientes, com os olhos fechados e os mostraram-se promissores para uso em pacientes anestesiados. / Intraoperative neurophysiological monitoring (MNIO) is a methodology that combines different neurophysiological tests for simultaneous or alternating in the same patient during the surgical procedure can evaluate the clinical neurophysiology in three fields: electroencephalography (EEG), electromyography (EMG) and evoked potential (EP). The visual evoked potential (VEP) evaluates the function and integrity of cortical and subcortical structures of the visual pathway. This test is performed daily in neurophysiology laboratories, helping the clarifying of different types of bouts vision, whether by disease or trauma. This study aimed to develop a prototype for monitoring the VEP to assess the integrity of the visual pathways during neurophysiological surgeries. Several steps were performed for the prototype ocular device: computer simulation; study of the characteristics of the human eye; rapid prototyping; Biopolymer characterization of polylactic acid (PLA) used in prototyping; development of an ocular device prototype with integrated lighting system and VEP achievement tests with the prototype. The PLA used in the prototype for the development of parts of the device eye was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), optical microscopy (OM), Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS), these results showed that the rapid prototyping did not change the physical-chemical and morphological PLA. The computer simulation provides appropriate parameters for the development of ocular device that allowed greater efficiency in the assembly of electronic circuit. The results of ENP tests were performed in different patients, with closed eyes and have shown promise for use in anesthetized patients.

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