Morphological changes, genetic modifications, and cell functional alterations are not always parallel. Therefore, assessment of skeletal muscle function is an integral part of the etiological approach. The general objective of this thesis was to look into the cellular and molecular events occurring in skeletal muscle contraction in healthy and diseased condition, using a single fiber preparation and a single fiber in vitro motility assay, in an attempt to approach the underlying mechanisms from different physiological angles. In a body size related muscle contractility study, scaling of actin filament sliding speed and its temperature sensitivity has been investigated in mammals covering a 5,500-fold difference in body mass. A profound temperature dependence of actin filament sliding speed over myosin head was demonstrated irrespective of MyHC isoform expression and species. However, the expected body size related scaling within orthologus myosin isoforms between species failed to be maintained at any temperature over 5,500-fold range in body mass, with the larger species frequently having faster in vitro motility speeds than the smaller species. This suggest that apart from the MyHC iso-form expression, other factors such as thin filament proteins and myofilament lattice spacing, may contribute to the scaling related regulation of skeletal muscle contractility. A study of a novel R133W β-tropomyosin mutation on regulation of skeletal muscle contraction in the skinned single fiber prepration and single fiber in vitro motility assay suggested that the mutation induced alteration in myosin-actin kinetics causing a reduced number of myosin molecules in the strong actin binding state, resulting in overall muscle weakness in the absence of muscle wasting. A study on a type IIa MyHC isoform missense mutation at the motor protein level demonstrated a significant negative effect on the function of the IIa MyHC isoform while other myosin isoforms had normal function. This provides evidence that the pathogenesis of the MyHC IIa E706K myopathy involves defective function of the mutated myosin as well as alterations in the structural integrity of all muscle irrespective of MyHC isoform expression.

Previous studies performed on myelinated nerve fibers have shown that a high frequency alternating current stimulus can block impulse conduction. The current threshold at which block occurs increases as the blocking frequency increases. Cable models based on the Hodgkin-Huxley model are consistent with these results. Recent experimental studies on unmyelinated nerve have shown that at higher frequencies, the block threshold decreases. When the block threshold is plotted as a function of frequency the resulting graph is distinctly nonmonotonic. Currently, all published models do not explain this behavior and the physiological mechanisms that create it are unknown. This difference in myelinated vs. unmyelinated block thresholds at high frequencies could have numerous clinical applications, such as chronic pain management. A large body of literature has shown that the specific capacitance of biological tissue decreases at frequencies in the kHz range or higher. Prior research has shown that introducing a frequency-dependent capacitance (FDC) to the Hodgkin-Huxley model will attenuate the block threshold at higher frequencies, but not to the extent that was seen in the experiments. This model was limited by the methods used to solve its higher order partial differential equation. The purpose of this thesis project is to develop a numerically stable method of incorporating the FDC into the model and to examine its effect on block threshold. The final, modified model will also be compared to the original model to ensure that the fundamental characteristics of action potential propagation remain unchanged.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 95-102). Also available for download via the World Wide Web; free to University of Oregon users.

In many behavioral paradigms, repeated exposures to a particular stimulus or event results in lower immediate early gene (IEG) expression. First, it was investigated if a similar reduction in IEG expression in the brain areas controlling male sexual behaviors would be observed after repeated copulation experiences in male Japanese quail. The results showed that IEG expression, as assessed by egr-1 immunoreactivity, did not increase in the POM, the BST, or the PAG after a copulation episode in highly sexually experienced subjects. One possibility was that the pattern of initial elevation of neuronal activity during the early trials of sexual interactions and the lack of increase in IEG expression later was associated with the novelty of sexual stimuli. While early exposures to certain stimuli constitute a new learning experience, the significance of such exposures would be lower as the level of experience increases. It was hypothesized that the introduction of a novel stimulus would increase the IEG expression in the POM, the BST and the PAG of experienced subjects. To evaluate this prediction, subjects were tested to see if they learn to respond to females decorated with distinct novel artificial cues after repeated exposures. The results showed that control subjects that did not have sexual experiences with decorated females discriminate against such females and directed their responses to normal females. Trained subjects did not show such preferences and responded to both types of females. In the next experiment, contrary to the prediction, no increase in IEG expression was observed after the introduction of the novel stimulus. This might be due to lower sexual motivation in subjects exposed to novel females. Effects of sexual experience were also tested in the catecholaminergic system. It was hypothesized that TH innervation in the POM and the BST would increase as a result of sexual experience. IEG expression in the catecholaminergic areas was predicted to be lower after repeated sexual experiences. The results showed no effect of experience in either tyrosine hydroxylase (TH) innervation, nor TH-egr-1 colocalization. These findings suggest that experience-related changes in male sexual behavior may be mediated by a different neurotransmitter system. / text

The Drosophila dorsal group gene pipe provides the crucial link that transmits dorsal-ventral (DV) polarity information from the ovary to the embryo. Females homozygous for mutations in pipe produce dorsalized embryos. pipe encodes ten protein isoforms with amino acid sequence similarity to vertebrate glycosaminoglycan 2-O-sulfotransferases, suggesting that Pipe functions by modifying a carbohydrate-bearing molecule that controls embryonic DV patterning. Two major components of my project have been to examine the functional specificities of different Pipe isoforms and to identify Pipe's enzymatic substrate and learn how it participates in DV pattern formation. I have used two approaches to investigate whether the various Pipe isoforms share the same functional specificities. In one approach, I expressed each isoform in the follicle cells and found that the expression of only one of them was able to rescue the pipe mutant phenotype or ventralize progeny embryos. In a second set of transgenic studies, three of the other isoforms were individually shown to restore the production of a pipe-dependent sulfated epitope when expressed in the salivary glands of otherwise pipe null mutant embryos. These data indicate that distinct functional specificities are associated with the various Pipe protein isoforms. In addition, these studies allowed me to determine that embryos from females lacking endogenous pipe expression nevertheless retain polarity along their dorsal-ventral axis, suggesting the existence of a second polarizing signal in addition to the ventral transcription of pipe. To identify Pipe’s substrate, I developed a technique for metabolic labeling which enabled me to identify a molecule exhibiting Pipe-dependent sulfation. This molecule was identified as the protein Vitelline Membrane-Like (VML), a putative component of the vitelline membrane layer of the eggshell. The involvement of VML in dorsalventral patterning was demonstrated on the basis of the enhancing effects of a vml mutation on the severity of dorsalization of embryos from females of a sensitized genetic background. Thus, VML represents a bona fide substrate of Pipe that participates in the establishment of dorsal-ventral polarity. In these studies I was also able to show Pipedependent sulfation of other vitelline membrane components which may also influence embryonic dorsal-ventral patterning. / text

No description available.

Obstetric trauma is a major cause of faecal incontinence in women resulting in considerable social disability. Until recently the development of faecal incontinence has been attributed largely to damage to the pelvic nerves. However the advent of anal endosonography has added a new dimension to our understanding of the pathophysiology of faecal incontinence. In this thesis, gross dissection and histological studies of 19 anorectal specimens was performed to demonstrate the normal anatomy. Simultaneous dissection and sonography of the anorectum (14 in vivo and 12 in vitro studies) has clarified the normal sonographic anatomy of the anal sphincters. Anal endosonography was performed in 114 healthy volunteers to demonstrate gender differences in anal sphincter anatomy. A prospective study of 12 patients undergoing secondary sphincter repair and 15 patients undergoing lateral internal anal sphincterotomy has validated the appearance of sonographic sphincter defects. A new technique of demonstrating the anal sphincters at rest using vaginal endosonography has been demonstrated in 20 women. A prospective study of 202 pregnant women using anal endosonography and neurophysiological tests has demonstrated that 35% of primigravidae (13% symptomatic) and 44% of multigravidae (23% symptomatic) develop occult anal sphincter defects during vaginal delivery. Although pudendal nerve damage can be identified in 16% of women 6 weeks after delivery, in the majority this recovers with time. Forceps delivery was identified as the single independent variable associated with sphincter damage although damage was also sustained in the absence of instrumental delivery. In a separate study of 50 women who sustained a recognized third degree tear 47% were found to be symptomatic despite a primary sphincter repair. In 85% of these women persistent anal sphincter defects were identified sonographically. In a further study of 43 women who had an instrumental delivery (17 vacuum and 26 forceps) anal sphincter defects were identified in 81% (38% symptomatic) of women who were delivered by forceps compared to 12% (21% symptomatic) delivered by the vacuum extractor. One hundred and fifty doctors and midwives were interviewed to assess their knowledge and training in perineal anatomy and repair. There was a clear deficiency in knowledge and inconsistencies in classification of third degree tears were apparent highlighting the need for more focused training in perineal anatomy and repair. / Thesis (Ph.D.)-University of Natal, 1994.

Rhodnius prolixus, the principal Chagas disease vector, requires a blood meal to complete its moult cycle into the next stage. Allatotropins (ATs), a family of peptides first isolated from Manduca sexta, have been shown to regulate the biosynthesis of juvenile hormone, an insect growth and development hormone; however, ATs, being multimodal peptides, also exhibit myotropic effects on some insect visceral muscles. Here, this AT family of peptides has been examined in R. prolixus. Genomic analysis revealed a cDNA fragment of 973bp encoding one mature amidated AT tridecapeptide (Rhopr-AT) with high transcript levels observed, via RT-PCR, in the central nervous system (CNS) and pool of fat body and trachea. AT-like immunoreactive neurons were found throughout the CNS and AT-like immunoreactive processes were present on some peripheral tissues. Bioassays using hindgut and dorsal vessel contraction, however, failed to demonstrate any effect of Rhopr-AT on these tissues. Future work will examine the effects of Rhopr-AT on JH production.

Rhodnius prolixus, the principal Chagas disease vector, requires a blood meal to complete its moult cycle into the next stage. Allatotropins (ATs), a family of peptides first isolated from Manduca sexta, have been shown to regulate the biosynthesis of juvenile hormone, an insect growth and development hormone; however, ATs, being multimodal peptides, also exhibit myotropic effects on some insect visceral muscles. Here, this AT family of peptides has been examined in R. prolixus. Genomic analysis revealed a cDNA fragment of 973bp encoding one mature amidated AT tridecapeptide (Rhopr-AT) with high transcript levels observed, via RT-PCR, in the central nervous system (CNS) and pool of fat body and trachea. AT-like immunoreactive neurons were found throughout the CNS and AT-like immunoreactive processes were present on some peripheral tissues. Bioassays using hindgut and dorsal vessel contraction, however, failed to demonstrate any effect of Rhopr-AT on these tissues. Future work will examine the effects of Rhopr-AT on JH production.

For decades electrophysiologists have recorded and characterized the biophysical properties of a rich diversity of neuron types. This diversity of neuron types is critical for generating functionally important patterns of brain activity and implementing neural computations. In this thesis, I developed computational methods towards quantifying neuron diversity and applied these methods for understanding the functional implications of within-type neuron variability and across-type neuron diversity. First, I developed a means for defining the functional role of differences among neurons of the same type. Namely, I adapted statistical neuron models, termed generalized linear models, to precisely capture how the membranes of individual olfactory bulb mitral cells transform afferent stimuli to spiking responses. I then used computational simulations to construct virtual populations of biophysically variable mitral cells to study the functional implications of within-type neuron variability. I demonstrate that an intermediate amount of intrinsic variability enhances coding of noisy afferent stimuli by groups of biophysically variable mitral cells. These results suggest that within-type neuron variability, long considered to be a disadvantageous consequence of biological imprecision, may serve a functional role in the brain. Second, I developed a methodology for quantifying the rich electrophysiological diversity across the majority of the neuron types throughout the mammalian brain. Using semi-automated text-mining, I built a database, Neuro- Electro, of neuron type specific biophysical properties extracted from the primary research literature. This data is available at http://neuroelectro.org, which provides a publicly accessible interface where this information can be viewed. Though the extracted physiological data is highly variable across studies, I demonstrate that knowledge of article-specific experimental conditions can significantly explain the observed variance. By applying simple analyses to the dataset, I find that there exist 5-7 major neuron super-classes which segregate on the basis of known functional roles. Moreover, by integrating the NeuroElectro dataset with brain-wide gene expression data from the Allen Brain Atlas, I show that biophysically-based neuron classes correlate highly with patterns of gene expression among voltage gated ion channels and neurotransmitters. Furthermore, this work lays the conceptual and methodological foundations for substantially enhanced data sharing in neurophysiological investigations in the future.