Discrete electrical stimulation of the organum vasculosum of the lamina terminalis (OVLT) produces sympathetically-mediated increases in peripheral resistance and arterial blood pressure (ABP). Since efferent fibers from the lamina terminalis innervate the kidney through polysynaptic connections, the present study determined whether electrical stimulation of the OVLT increased sympathetic outflow to the kidney. In anesthetized male, Sprague-Dawley rats (n=5) electrical stimulation of OVLT neurons produced frequency and current intensity dependent increases in renal sympathetic nerve activity (RSNA) and ABP that were abolished by ganglionic blockade with the nicotinic antagonist chlorisondamine (5mg/kg,i.v.). Since neurons from the OVLT terminate within the hypothalamic paraventricular nucleus (PVH), the present study also determined whether these connections mediate a portion of sympathetic and pressor responses to electrical stimulation of the OVLT. Bilateral inhibition of the PVH with the GABAA agonist muscimol (5mM/100nl) significantly attenuated the increase in ABP at all frequencies and current intensities. Spike-triggered averaging of RSNA revealed that PVH inhibition significantly blunted the RSNA responses to OVLT stimulation at 100, 200, but not 400andamp;igrave;A. The present findings indicate that electrical stimulation of the OVLT increases RSNA and ABP and that these responses are partially mediated by the tonic activity of PVH neurons.

Small lowland agricultural systems promote conditions where benthic biological communities can thrive. These biogeochemical processes have significant impacts on terrestrial ecosystem processes including POC flux and fate, nutrient balances, water quality budges, and aquatic biological functioning. Limited information is available on coupled biological and hydrologic processes in fluvial systems. This study investigates the mixture of biological and hydrologic processes in the benthic layer in order to understand POC cycling in the South Elkhorn system. Further, comprehensive modeling of POC flux in lowland systems has not been performed previously and the behavior of potentially controlling variables, such as hydrologic forcing and seasonal temperature regimes, is not well understood. Conceptual hydraulic and sediment transport models were simulated for the South Elkhorn. Based on data and model results it was concluded that during a hydrologic event, upland and bank sources produce high variability of POC sources. Likewise, over time, the density of hydrologic events influenced accrual of benthic algal biomass in the POC pool. Environmental variables such as temperature and light availability drove seasonal variations of POC in the streambed. Based on model estimates, around 0.29 metric tCkm-2yr-1 of POC is flushed from the system annually with 13 % coming from autochthonous algae.

Glaucoma is a leading cause of irreversible blindness worldwide. Primary Open Angle Glaucoma is the most common form of the disease and can be characterized by the slow and irreversible apoptotic death of retinal ganglion cells, a unique optic nerve neuropathy resulting in loss of vision. Increased intra-ocular pressure is known to be a leading risk-factor for glaucoma, and lowering IOP is currently the only evidence based method for the clinical management of the disease. However the exact mechanism by which an elevated IOP leads to the death of the retinal ganglion cells is still poorly understood. By using previous finite element models of glaucoma to quantify the biomechanical environment within the optic nerve head we have built human primary cell culture models in an attempt to replicate aspects of early glaucomatous optic neuropathy. In these models we mimic the in vivo biomechanical environment in the lamina cribrosa by growing human optic nerve head astrocytes and lamina cribrosa cells on compliant substrates and subjecting the cells to deformation. Specifically, a global protein scan using isobaric tags for relative and absolute quantitation (iTRAQ) was performed on all the experiments to identify potential biomarkers for glaucoma. A secondary analysis using enzyme-linked immunosorbent assay (ELISA) identified extracellular proteins of interest. Over 520 proteins were identified in response to biomechnical strain from both cell types. Many of these proteins centred on TGF-, p53 and TNF, which have previously been shown to play a role in the pathogenesis of glaucoma. Proteins found in astrocytes were astrocytic phosphoprotein (PEA15), UDP-glucose dehydrogenase (UGDH), and annexin A4 (ANXA4). LC proteins were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66) and Eukaryotic translation initiation factor 5A (eIF-5A). These proteomic results will enable a series of functional studies looking into the role select markers play in ONH glial cell activation, a process still not well understood. Candidates for this work will be prioritized based on novelty and relevance to mechanisms of cellular stress and death. We hypothesize that study of these molecular pathways will provide insight into this process, as well as improve our understanding of how glial activation contributes to the development of glaucomatous optic neuropathy.

Glaucoma is a leading cause of irreversible blindness worldwide. Primary Open Angle Glaucoma is the most common form of the disease and can be characterized by the slow and irreversible apoptotic death of retinal ganglion cells, a unique optic nerve neuropathy resulting in loss of vision. Increased intra-ocular pressure is known to be a leading risk-factor for glaucoma, and lowering IOP is currently the only evidence based method for the clinical management of the disease. However the exact mechanism by which an elevated IOP leads to the death of the retinal ganglion cells is still poorly understood. By using previous finite element models of glaucoma to quantify the biomechanical environment within the optic nerve head we have built human primary cell culture models in an attempt to replicate aspects of early glaucomatous optic neuropathy. In these models we mimic the in vivo biomechanical environment in the lamina cribrosa by growing human optic nerve head astrocytes and lamina cribrosa cells on compliant substrates and subjecting the cells to deformation. Specifically, a global protein scan using isobaric tags for relative and absolute quantitation (iTRAQ) was performed on all the experiments to identify potential biomarkers for glaucoma. A secondary analysis using enzyme-linked immunosorbent assay (ELISA) identified extracellular proteins of interest. Over 520 proteins were identified in response to biomechnical strain from both cell types. Many of these proteins centred on TGF-, p53 and TNF, which have previously been shown to play a role in the pathogenesis of glaucoma. Proteins found in astrocytes were astrocytic phosphoprotein (PEA15), UDP-glucose dehydrogenase (UGDH), and annexin A4 (ANXA4). LC proteins were bcl-2-associated athanogene 5 (BAG5), nucleolar protein 66 (NO66) and Eukaryotic translation initiation factor 5A (eIF-5A). These proteomic results will enable a series of functional studies looking into the role select markers play in ONH glial cell activation, a process still not well understood. Candidates for this work will be prioritized based on novelty and relevance to mechanisms of cellular stress and death. We hypothesize that study of these molecular pathways will provide insight into this process, as well as improve our understanding of how glial activation contributes to the development of glaucomatous optic neuropathy.

As pregas vocais (PV) são estruturas únicas, altamente especializadas na vibração para a produção sonora. Em grande parte, decorrente da estruturação em camadas da lâmina própria (LP). Essa estruturação não está presente ao nascimento, somente após anos de uso vibratório e fonatório da PV é que a LP está finalmente estruturada. As pregas ventriculares (PVT) não são, habitualmente, estruturas vibratórias na produção sonora, e possuem a LP menos organizada em estratos, sendo menos especializadas para a vibração. Até o presente momento, não se tem conhecimento do que ocorre com os constituintes da LP de PVTs de pessoas que as utilizam como fonte produtora de voz. No presente estudo, foram comparados os constituintes colágenos e as fibras elásticas da LP de PVTs de indivíduos que as utilizam como principal fonte vibratória na produção de voz com o grupo controle. Foram selecionados seis indivíduos que utilizavam pelo menos uma das PVTs como fonte de vibração para a produção sonora por ao menos seis anos. Delas, colheu-se pequeno fragmento (0,5 cm2), que após processamento histológico, as fibras colágenas foram coradas com Picrosirus Red e as fibras elásticas com Weigert resorcina-fucsina. Foram obtidas 54 imagens da camada mais superficial da LP de cada PVT para cada coloração. Após a aquisição das imagens, as fibras colágenas tipo I e tipo III, colágenas totais e fibras elásticas foram quantificadas utilizando-se o software Image-Pro Plus, e comparadas com as PVTs dos controles. A análise estatística foi realizada por meio do teste T de Student para amostras não pareadas. A porcentagem de colágeno total na camada mais superficial da LP de PVT utilizada como fonte vibratória para a produção de som foi significativamente maior em relação aos controles. O mesmo ocorreu com a quantidade de colágeno tipo I. Não houve diferenças na quantidade de colágeno tipo III e de fibras elásticas. Como conclusão, pode-se afirmar que a utilização da PVT como fonte vibratória produtora de som leva ao aumento da quantidade de fibras colágenas totais e do tipo I na camada mais superficial da sua LP / The vocal folds (VF) are unique structures, highly specialized in vibrating for sound production. This specialization is mainly due to a layered structure of the lamina propria (LP). This layered structure is not present at birth, and develops only after a several years of phonation. The LP of the mature vocal fold consists of three layers. The ventricular folds (VTF) are not originally vibrating structures for sound production, and in its LP the layers are poorly organized. It is not known what happens to the constituents of the LP in the VTF in subjects that use VTF vibration as a source of voice production. In the present study, the distribution and quantity of collagen and elastic fibers of the lamina propria from VTF of patients that use it as the main source of vibration for voice production were compared with the VTF from control subjects. Six individuals that used at least one of the VTF as source of vibration for sound production for minimum of six years were selected. A small fragment of VTF (0.5 cm2) used as vibration source of sound production was collected from each subject. The samples were processed for histological analysis. Collagen fibers were stained with Picrosirus Red and elastic fibers were stained with Weigert\'s Resorcin-Fuchsin. A total of 54 images were obtained from the superficial layer of the LP from each VTF for each stain. After image acquisition, collagen type I, III, total collagen and elastic fibers were quantified and compared with the VTF from the control group. Quantification was done using Image-Pro Plus software. Statistics were performed using an unpaired Student T test. The amount of total collagen in the most superficial layer of LP when the VTF was used as the source of vibration for the production of sound was significantly higher when compared to controls. The same result was seen for the amount of type I collagen in both groups. There was no difference in the quantity of type III collagen and elastic fibers between the two groups. Vibration of the VTF as a source of sound, for at least six years, leads to an increase in the amount of total collagen fibers and an increase in type I collagen, but does not increase the amount of type III collagen and elastic fibers in the most superficial layer of LP. These results may help elucidate the unique development of the lamina propria of the vocal fold

Logging of tropical forests is increasing worldwide. Logging alters the forest conditions such as temperature, soil water content and litter input into the soil. This study explored how soil invertebrate activity in Borneo differs between pristine forests and two secondary forests, with 10 and 40 years of recovery time since the last logging. To measure the soil fauna feeding activity, the bait lamina stick method was applied. The study was conducted in Sabah, Malaysian Borneo, during April and May 2019. 33 forest plots were examined with ten lamina sticks placed in each of the three replicas per forest plot. The sticks were kept in the soil for four weeks before being removed. Upon removal, the soil invertebrate activity was determined by assessing how many holes of the bait lamina sticks were eaten and at what depth. The activity was related to the above-ground carbon density (ACD, a density measure for amount of above-ground carbon), as well as depth-specific activity in the different plots. Moreover, further relationships with the invertebrate activity and environmental conditions such as cumulative throughfall during the study time as well as the soil water content were studied. The results showed that the soil activity slightly decreased with increased ACD, but no statistical significance was found. This study also suggests that the activity was higher in the upper 0-5cm of the soil than in the lower 5-10 cm. Lastly, the results showed that the activity was highest in the forest with the shortest recovery time (10 years). This implies that it might be possible to regain the original soil activity since the activity of the 40-year-old forest was closer to the pristine forest than that of the 10- year-old forest.

Philosophiae Doctor - PhD / Histological observations revealed that oral epithelium originated from a single ectodermal layer. As the ectoderm grew so it differentiated into squamous epithelium. The first features of squamous differentiation were noticed at 8 weeks in utero in areas where keratinized mucosae were developing, and these were the changing of cuboidal to cylindrical basal cells and the subsequent growth of prickle cells from these cylindrical basal cells. The prickle cells merged with the existing primitive cells and at no stage could a separate squamous epithelial layer I such as the stratum tritermedium of the epidermis I be observed inside the mouth. At 12 weeks in utero squamous differentiation had reached a stage where acidophilic layers appeared in certain regions on the epithelial layer. The time of appearance of these layers varied from case to case. At this stage most of the primitive characteristics had disappeared from the keratinizing epithelium. Unlike the periderm of the skin which was shed into the amniotic fluid, shedding of primitive epithelial cells from the keratinizing squamous epithelium was not noticeable. Thence, the growth of keratinizing epithelium was followed by an increase of acidophilic layers, the appearance of keratohyaline granules in cells and, in some instances, full keratinization. The latter I however I was almost exclusively confined to the vermilion border of the lip. The squamous epithelium of the lining mucosa, which is unkeratinized I developed at a much slower tempo. It retained its cuboidal-shaped basal cells and the primitive features of the overlying cells were lost only at about 4- 5 months in utero I when squamous differentiation set in. At no stage was the squamous differentiation a prominent feature. At junctions between keratinized and unkeratinized epithelia and epidermis the epithelium exhibited features of both types of epithelia that were being joined. This was especially noticeable at the junction between vermilion epithelium and epidermis, where part of the vermilion epithelium displayed a prominent intermediate type of layer. Similarly, acidophilic layers of keratinizing epithelium merged imperceptibly with the walls of cells of unkeratinizing epithelium, creating a small region of an unkeratinizing type of epithelium with keratinized cells. Thus the development of the oral epithelium is through differentiation and renewal of epithelial cells: the ectodermal layer developes into an epithelial layer which is recognised by its squamous appearance. The subsequent growth is by constant renewal of this differentiated epithelium. The pattern of epithelial development I the appearance of the junctional epithelia and the manner in which acidophilic layers merge with unkeratinized epithelial cells I indicate a unity between these epithelia. According to these developmental features, the epithelium of the mouth and epidermis can be classified into less differentiated and better differentiated, but with a commonbackground for these epithelia. When the formation and the established appearance of keratin in the mouth and on the skin was compared histologically I ultrastructurally and histochemically I a unity between these features became apparent. Ultrastructurally it appeared that keratin consisted basically of 2 cytoplasmic constituents: tonofilaments and a fine granular substance. The tonofilaments were gathered at first into bundles and then broken up into finer tonofibrils. These finer fibrils mixed with a granular ground substance to form a homogenous granular filamentous material. This product can be regarded as a pre-keratin. With the addition of a keratohyaline layer to the process I keratin was formed, Apart from the keratohyaline granules several additional changes took place in cells concerned in this process I whether keratin was formed or not. These changes were flattening of cells, extensive interdigitation between cell walls, disappearance of micro-villi I loss of structure in desmosomes I thickening of cell walls and the disappearance of glycogen from cells. Some of these features were displayed in each of the types of epithelium examined here.

La forme du noyau peut varier significativement au cours du développement ou lors de processus pathologiques en raison des forces mécaniques émanant du microenvironnement ou générées par le cytosquelette. L’impact de la morphologie nucléaire sur la machinerie transcriptionnelle n’est cependant pas connu. En utilisant plusieurs approches afin de manipuler la morphologie nucléaire, nous avons observé que des changements de forme de l’enveloppe nucléaire régulent l’activité de AP1 et TEAD. Nous avons montré que l’aplatissement du noyau augmente la phosphorylation de c-Jun et la translocation de YAP, conduisant à une augmentation de la transcription des gènes cibles de AP1 et TEAD. Nous avons également observé que l’aplatissement du noyau se produit au cours du cycle cellulaire et favorise la prolifération via l’activation de TEAD et AP1 qui stimulent la progression de la phase G1 à la phase S. / .The shape of the cell nucleus can vary considerably during developmental and pathological processes as a consequence of the mechanical forces emanating from the microenvironment or generated by the cytoskeleton. However the impact of nuclear morphology on the transcriptional machinery is not known. Using a combination of tools to manipulate the nuclear morphology, we observed that changes in nuclear shape regulate the activity of AP1 and TEAD. We showed that nuclear flattening increases c-Jun phosphorylation and YAP nuclear translocation, leading to transcriptional induction of AP1 and TEAD-target genes. Surprisingly, we found that nuclear compression is necessary and sufficient to mediate c-Jun and YAP activation in response to cell- generated contractility or cell spreading. We additionally observed that nuclear flattening occurs during the cell cycle and promotes proliferation via TEAD and AP1- dependent G1 to S progression.

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A deeper understanding of human development and disease is made possible partly through the study of genetically modified model organisms, such as the common mouse (Mus musculus). By genetically modifying such model organisms, scientists can activate, deactivate, or highlight particular characteristics. A genetically modified animal is generated by adding exogenous (foreign) genetic material to one or more embryonic cells at their earliest stages of development. Frequently, this exogenous genetic material consists of specially engineered DNA, which is introduced into a fertilized egg cell (zygote). When successfully introduced into the zygote, the exogenous DNA will be incorporated into the cell's own genome, and the animal that develops from the zygote will exhibit the genetic modification in all of its cells. The current devices and methods for generating genetically modified animals are inefficient, and/or difficult to use. The most common and efficient method for inserting new DNA into zygotes is by directly injecting a DNA solution through a tiny glass tube into the cell in a process called microinjection. Unfortunately, microinjection is quite inefficient (success rates are commonly between 1 and 5%), but often it is the only method for inserting DNA into eggs, zygotes, or early stage embryos. This thesis presents the design and testing of a micrometer sale, pumpless microelectromechanical system (MEMS) nanoinjector. Rather than use pumps and capillaries, the nanoinjector employs electrostatic charges to attract and repel DNA onto and off of the surface of a solid lance. The nanoinjector also includes a mechanical system for constraining the target cells during injection. Initial testing indicates the nanoinjector does not decrease cell viability, and it has a very high initial success rate (up to 90%). With the addition of an on-chip actuator, the nanoinjector could be packaged as an inexpensive, fully automated system, enabling efficient, high volume genetic modification of developing animals. Such a device would greatly increase the ease and speed of generating the model organisms needed to study such critical diseases such as Alzheimer's disease, cancer, and diabetes.