[Truncared abstract] Introduction: The assessment of any therapy requires not only an understanding of how that therapy works but also how it affects health related quality of life (HRQOL). Botulinum Toxin A(BoNT-A) therapy for upper limb spasticity management in children with hemiplegic cerebral palsy(CP) is currently under trial. Despite its use for over a decade for lower limb spasticity, little is known about the mechanisms involved in improving motor function and the effect on the child and their familys HRQOL. Both central and peripheral mechanisms are hypothesised[1]. Whilst evidence of improved quality of movement and ability to perform tasks is emerging[2-4], this cannot be directly correlated with an improvement in HRQOL. In addition, the traditional method of assessing child HRQOL by parent proxy reports has come under question[5, 6]. The World Health Organisation now recommends the collection of both parent proxy and child self-reports[7]. Aims: 1. Investigate the corticomotor projections to the upper limb in school aged children with hemiplegic CP and the changes that occur with BoNT-A therapy by transcranial magnetic stimulation (TMS). 2. Investigate the effect of upper limb BoNT-A therapy on HRQOL of school aged children with hemiplegic CP by completion of the PedsQL 4.0 Generic Core Scales and 3.0 CP Module. 3. Determine the concordance between Child Self-Report and Parent Proxy Report scores for the PedsQL 4.0 Generic Core Scales and 3.0 CP Module. 4. Determine the concordance between PedsQL scores and function as assessed by the Melbourne Assessment of Unilateral Upper Limb Function (MUUL). Methods: Design: Prospective randomised pilot study. Setting: Department of Paediatric Rehabilitation, Princess Margaret Hospital, and Centre for Neurological and Neuromuscular Disorders, Perth. Participants: 22 school aged children with hemiplegic CP aged 7yr 0mth-13yr 11mth (12 treatment, 10 control). 3 Treatment: One episode BoNT-A injections (dose 1-2U/kg/muscle) into the upper limb for treatment group. The control group received usual care. ... Conclusion: This pilot study provides preliminary evidence of the effects of upper limb BoNT-A therapy at both a central physiological and a broader quality of life level in school aged children with hemiplegic CP. At a central level, corticomotor pathway reorganisation occurs in the setting of BoNT-A. However the reorganisation is not limited to the affected side pathways suggesting a systemic BoNT-A effect or developmental changes. Similarly, in this pilot study, there was no statistically significant effect of upper limb BoNT-A on the childs HRQOL as assessed by the PedsQL although positive trends were observed 4 for a number of physical and psychosocial domains. The collection of both child self-report and parent proxy reports when assessing HRQOL is recommended, and function needs to be assessed independently. Larger studies across the broader CP population, the design of CP specific HRQOL tools appropriate for use in the higher functioning CP cohort, and alternative better tolerated methods of investigating the motor system in children with movement disorders are recommended.

Proteases play an indispensable role in medicine, serving both as drug targets and as therapeuticagents. Protease inhibitors are a key component of our antiviral arsenal, and are widely used to combat human immunodeficiency virus (HIV), hepatitis C virus (HCV), and, most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nirmatrelvir, an inhibitor of the 3-chymotrypsin like (3CL) protease essential for SARS-CoV-2 replication, was granted emergency use authorization in late 2021, formulated with ritonavir and sold under the brand name Paxlovid. Since then, Paxlovid use has become widespread, raising the possibility that nirmatrelvir resistance could emerge in circulating SARS-CoV-2. It is therefore important to understand how the 3CL protease might mutate to lose nirmatrelvir sensitivity so that circulating variants can be monitored for these mutations and future generations of inhibitors can be designed to prevent cross-resistance. The most widely used therapeutic proteases are the botulinum neurotoxins (BoNTs),which are effective in the treatment of a wide array of movement, pain, and autonomic disorders. These toxins exert their therapeutic effect by cleaving members of the SNARE protein family inside neuronal cytosol, preventing neurotransmitter release. Much work has been dedicated to engineering members of the BoNT family to extend their therapeutic utility, including altering receptor tropism, extending half-life, and modifying protease specificity. While significant progress has been made in each of these areas, the extent to which these proteases can be reprogrammed to target the proteins that cause human disease remains unexplored. In this dissertation, we employ diverse methods to study and direct protease evolutionand to facilitate protein engineering more broadly. To investigate the emergence of nirmatrelvir resistance, we passage SARS-CoV-2 in increasing concentrations of the drug and sequence the 3CL protease gene over time in resistant lineages. We then validate the observed 3CL protease mutations by incorporating them into recombinant SARS-CoV-2 and testing the nirmatrelvir sensitivity of the resulting viruses. We find that the development of nirmatrelvir resistance typically begins with the acquisition of a precursor mutation such as T21I, P252L, or T304I, which confers a low level of resistance and enables strong resistance conferring mutations, such as E166V, to emerge without imposing a significant fitness cost. To explore the programmability of the BoNT proteases, we engineer type E (BoNT/E) to cleave proteins involved in neurodegenerative disease. To accomplish this, we employ targeted mutation based on a structural model as well as the continuous evolution platform known as OrthoRep, selecting for variants that cleave the desired substrates with a circuit that links protease activity to the growth of a Saccharomyces cerevisiae (yeast) cellular chassis. We first use this approach to target ATXN3, the protein whose aggregation causes type 3 spinocerebellar ataxia (SCA3). We then profile the substrate specificities of the BoNT/E variants that emerged during our ATXN3 engineering, identifying patterns that can inform the selection of new targets. Based on one of these patterns, we generate a new BoNT/E variant capable of cleaving TDP-43, a protein implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Finally, to facilitate future protein engineering campaigns, we develop a machine learning pipeline that uses deep mutational scanning (DMS) data to model a protein’s fitness landscape and predict optimized variants with user-defined constraints. We name this pipeline OptiProt, and we demonstrate its utility by applying it to the human chaperone DNAJB6, which rescues cellular toxicity in a yeast model of ALS. In this context, we probe OptiProt’s engineering capabilities with a range of challenges. We first predict hyper-functional variants with up to 50 mutations. We then restore activity to variants harboring one of two known loss-offunction mutations. Finally, to demonstrate the OptiProt’s amenability to complex engineering constraints, we restore activity to a variant harboring a deleterious mutation while simultaneously mutating a set of highly conserved residues. Altogether, the work presented in this dissertation highlights the value of laboratory protease evolution in two clinically relevant applications and provides a tool that can facilitate future work in this field.

Thesis (MScPhysio (Physiotherapy))--Stellenbosch University, 2008. / Cerebral palsy (CP) is a common neurological condition seen in children which results in childhood disability. Damage to the developing brain results in abnormal muscle tone and decreased force generation, which leads to loss of independent function. Previous studies investigating interventions targeting the typical equinus gait pattern seen in spastic CP have reported inconclusive and widespread outcomes. Objectives The objectives of the study were to determine (1) the effect of functional electrical stimulation (FES) after a single botulinum toxin injection into the triceps surae muscle as a functional orthosis on various gait parameters and economy of movement; (2) caregivers’ perceptions of the impact of the intervention on their child’s function and participation, and (3) optimal timing intervals for introducing FES after a botulinum toxin injection. Method Single-subject research with a multiple baseline approach was conducted on five ambulant subjects (average age 5.1 years, SD=1.4) in the Cape Metropole with a dynamic equinus gait due to hemiplegic CP. Two-dimensional gait analysis, isometric dynamometry, Energy Expenditure Index (EEI), and a caregiver questionnaire were used to gather data on walking speed, ankle angles at initial contact of gait, isometric plantarand dorsiflexior muscle strength, energy expenditure during gait, as well as caregiver perception on participation changes. Statistical analysis was conducted by means of ANOVA tests and graphic data illustrations. Results A statistically significant pre- to post intervention (FES after botulinum toxin) change was found for plantarflexor muscle strength. This effect was partially maintained over the withdrawal phase. Caregivers felt the intervention to have a positive influence on their children’s walking speeds, as well as on age-appropriate function and participation. Selfselected walking speed, dorsiflexor muscle strength, and ankle angles at initial contact did not change significantly. A 32-day interval between between botulinum toxin and the FES programme resulted in the most pronounced improvements in terms of walking speed, EEI scores, and plantarflexor muscle strength. Conclusion FES to the lower limb, 32 days after botulinum toxin into the triceps surae, applied for 30 minutes per day, five times a week over a total of four weeks, seemed to improve selected gait parameters as well as caregiver perception of impact on function and activities of daily living. However, further research is needed.