Spinal muscular atrophy (SMA) is the most commonly inherited neurodegenerative disease that leads to infant mortality worldwide. There are no known cures for SMA, but small increase in protein levels of SMN can be beneficial. We have developed adenoviral (Ad) vectors that express a human transgene of SMN and have tested their safety in vitro. We have demonstrated that these viruses can effectively express the transgene following cell entry and that the levels are relative to the virus dose. The viruses do not appear to impact the health and function of the cells, and are capable of increasing the number of Gems. We also attempted to change the tropism of the viruses through fiber protein modifications in order to target muscles and motor neurons. Our results suggest that a therapy based on an Ad-SMN fiber-modified vector may ultimately be successful in treating patients of SMA.

The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

Compared with our knowledge concerning the responses to exercise of able-bodied athletes there is relatively little known about the exercise responses of athletes who have a spinal cord injury (SCI). The purpose of this thesis was to examine the physiological characteristics of endurance trained wheelchair athletes; to assess the importance of various physiological factors to the endurance performance of wheelchair athletes; to describe and examine the physiological and metabolic responses and performances of wheelchair athletes to endurance and brief high intensity exercise; and to assess the influence of level of SCI or wheelchair racing class on these responses to exercise. The mean peak oxygen uptake (V02 pk) achieved by the group of 27 wheelchair athletes during wheelchair treadmill exercise was 2.11 ±0.53 I. min-1. When the athletes were grouped according to the paralympic racing classification system (TK2; TK3; TK4) the V02 pk values were 1.28 ±0.16 l. min-1,2.10 ±0.43 I. min-1 and 2.32 ±0.52 I. min-1 respectively. The mean peak heart rates of these groups were 112 ±4 b. min-1,190 ±9 b. min-i, and 200 ±9 b. min-1 respectively. The relationship between V02 pk and endurance performance (r=-0.69; p<0.01) was not as high as found between % V02 pk utilised at a given submaximal propulsion speed (r=0.89; p<0.01) or a propulsion speed equivalent to a reference blood lactate concentration (BLA) of 4 mmol. l-1 (r=-0.87; p<0.01). During a 10 km treadmill trial the group of athletes maintained a speed equivalent to 78.4 ±13.6% V02 pk. The TK2 racing class appeared to exhibit lower heart rates and respiratory exchange ratio (R) values throughout the test than the other racing classes. During a one hour endurance test at 80% of top speed (TS), an exercise intensity meaningful to the wheelchair athlete, the group were working at 64.6 ±13.5% V02 pk. The responses observed were indicative of steady state exercise. Oxygen uptake and ventilation rate remained stable, there was no cardiovascular drift, there was a decrease in R value, BLA peaked after 15 minutes and then decreased throughout the remainder of the test. Blood glucose (BGL) concentrations remained similar to the value observed at rest throughout the test. In general, it was found that wheelchair athletes were able to maintain a propulsion speed equivalent 75% V02 pk for prolonged periods of time, irrespective of their level of SCI. There were no differences between the physiological and metabolic responses of the two paraplegic racing classes. The tetraplegic group appeared to exhibit lower heart rates, BLA, BGL and R values throughout the duration of the test. The concentrations of plasma free fatty acid, glycerol, ammonia and urea postexercise indicated a tendency towards higher values in the wheelchair racing class with the lowest lesion level athletes (TK4). During a 20 s high intensity exercise fixed work test there was greater metabolic activity in the TK2 racing class than the TK4 racing class. The metabolic responses of the TK3 racing class to this test were, in general, between these two groups. A 30 s arm sprint resulted in a decrease in power output of greater than 50% for each racing class. The mean power outputs (MPO) generated by the TK2, TK3 and TK4 racing classes were 100.2 ±21.7 W, 188.3 ±48.9 W and 247.2 ±40.3 W respectively. The physiological and metabolic responses showed a similar tendency. A 30 s test at 80% MPO showed that the metabolic responses of each racing class were similar. The results of the thesis suggest that the amount of muscle mass available for recruitment during exercise and the degree of disruption to the sympathetic nervous system play an important role in determining the responses to exercise and the racing performance of wheelchair athletes with a SCI.

Previous research has demonstrated that the spinal cord is capable of a simple form of instrumental learning. In this instrumental learning paradigm, rats typically receive a complete spinal transection at the second thoracic vertebra, and are tested 24 hours after surgery. Subjects that receive shock to a hind leg quickly learn to maintain the leg in a flexed position, which reduces net shock exposure (Grau et al., 1998). Prior studies have examined the mechanisms that mediate this learning, but little is known about how or where the consequences of learning are stored (memory). The goal of this dissertation proposal is to examine the neural modification(s) that preserve learned behavioral effects over time. It is clear that the central nervous system plays an essential role in instrumental learning. During the acquisition of instrumental learning, the connections between the peripheral nervous system (PNS) and the central nervous system must remain intact (Crown et al., 2002a). Acquisition is also disrupted by intrathecal application of pharmacological agents (lidocaine) that inhibit spinal reflexes (Crown et al., 2002a). The experiments outlined in this dissertation are motivated by an unexpected observation: while application of lidocaine to the spinal cord prior to training blocks acquisition of the instrumental response, inactivating spinal neurons has no effect on the maintenance of the instrumental response. These data suggest that, after the instrumental response is acquired, a peripheral component is capable of maintaining the instrumental response. Aim 1 examined how inhibiting the spinal cord influenced the maintenance of instrumental learning. Intrathecal lidocaine inhibited a spinal withdrawal reflex and instrumental learning, but did not affect the maintenance of the learned response. Expanding on these results, Aim 2 examined how disconnecting the PNS from the spinal cord would influence the maintenance of instrumental learning. If a PNS to spinal cord connection is needed for the maintenance of instrumental learning, then removing that connection by a sciatic transection should disrupt performance of the instrumental response. Together, the results of Aims 1 & 2 confirm that a peripheral alteration contributions to the maintenance of instrumental behavior. In Aim 3, I developed a procedure that would allow for drug delivery directly to the tibialis anterior muscle. If the neuromuscular junction is capable of influencing a spinal reflex, then blocking the neuromuscular junction with an antagonist (curare) should disrupt the acquisition and maintenance of the instrumental response. Based on the results of Aim 3, Aim 4 investigated how other pharmacological manipulations at the neuromuscular junction can influence the acquisition and maintenance of the instrumental response. Using glutamate receptor antagonists (CNQX and MK-801), I showed that glutamatergic signaling plays an essential role.

Thesis (MAppSc in Physiotherapy)--University of South Australia, 1995

Spinal cord injury can result in partial or complete loss of supraspinal control of sympathetic outflow below the lesion. Despite the importance of the sympathetic nervous system in autonomic dysreflexia and orthostatic hypotension, its integrity is not currently assessed in the clinical determination of lesion level - only motor and sensory pathways are examined. The aim of this thesis was to develop non-invasive means of assessing the integrity of sympathetic pathways following human spinal cord injury. Brief innocuous electrical stimuli applied to the forehead evoked cutaneous vasoconstrictor and sudomotor responses in the fingers and toes of able-bodied subjects, which were abolished by complete spinal lesions. Application of these same stimuli to the abdominal wall (below lesion) generated long-lasting cutaneous vasoconstriction (but not sweat release) and significant increases in blood pressure that accumulated with repeated stimuli. Moreover, the magnitude and duration of these spinal somatosympathetic reflexes did not depend on the number of impulses or duration of the sensory input, suggesting that only the initial part of the sensory barrage elicits reflex responses. This work has shown that cutaneous vascoconstriction provides a more robust measure of the integrity of sympathetic pathways than does sweat release. This was confirmed during natural stimulation of somatic afferents during vibroejaculation, which caused marked increases in blood pressure and marked cutaneous vasoconstriction but negligible sweat release below lesion. Muscle vasoconstrictor function below lesion was assessed by asking subjects to perform a maximal inspiratory breath-hold, which is known to cause a sustained activation of muscle vasoconstrictor neurones that counteracts the fall in blood pressure in able-bodied subjects. Blood pressure remained low in paraplegics and, especially, quadriplegics during this manouevre; importantly, heart rate showed a linear increase only in the spinal patients. In the absence of blood pressure measurements, the latter infers an interruption of descending muscle vasoconstrictor pathways. In conclusion, this thesis has demonstrated simple, non-invasive techniques that can be utilised to assess the function of the sympathetic nervous system in spinal cord injury, and highlighted the need to assess the injury in terms of the integrity of the sympathetic nervous system below the lesion level.

Spinal cord injury can result in partial or complete loss of supraspinal control of sympathetic outflow below the lesion. Despite the importance of the sympathetic nervous system in autonomic dysreflexia and orthostatic hypotension, its integrity is not currently assessed in the clinical determination of lesion level - only motor and sensory pathways are examined. The aim of this thesis was to develop non-invasive means of assessing the integrity of sympathetic pathways following human spinal cord injury. Brief innocuous electrical stimuli applied to the forehead evoked cutaneous vasoconstrictor and sudomotor responses in the fingers and toes of able-bodied subjects, which were abolished by complete spinal lesions. Application of these same stimuli to the abdominal wall (below lesion) generated long-lasting cutaneous vasoconstriction (but not sweat release) and significant increases in blood pressure that accumulated with repeated stimuli. Moreover, the magnitude and duration of these spinal somatosympathetic reflexes did not depend on the number of impulses or duration of the sensory input, suggesting that only the initial part of the sensory barrage elicits reflex responses. This work has shown that cutaneous vascoconstriction provides a more robust measure of the integrity of sympathetic pathways than does sweat release. This was confirmed during natural stimulation of somatic afferents during vibroejaculation, which caused marked increases in blood pressure and marked cutaneous vasoconstriction but negligible sweat release below lesion. Muscle vasoconstrictor function below lesion was assessed by asking subjects to perform a maximal inspiratory breath-hold, which is known to cause a sustained activation of muscle vasoconstrictor neurones that counteracts the fall in blood pressure in able-bodied subjects. Blood pressure remained low in paraplegics and, especially, quadriplegics during this manouevre; importantly, heart rate showed a linear increase only in the spinal patients. In the absence of blood pressure measurements, the latter infers an interruption of descending muscle vasoconstrictor pathways. In conclusion, this thesis has demonstrated simple, non-invasive techniques that can be utilised to assess the function of the sympathetic nervous system in spinal cord injury, and highlighted the need to assess the injury in terms of the integrity of the sympathetic nervous system below the lesion level.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 27, 2009) Vita. Includes bibliographical references.