No description available.

No description available.

Huntington’s disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HTT gene, which causes an expansion in the polyglutamine tract of the huntingtin protein. In 2018, the cryo-EM structure of the 350 kDa protein huntingtin (Htt) in complex with huntingtin associated protein of 40 kDa (HAP40) was solved, which demonstrated that huntingtin had to be co-translated and complexed with HAP40 to retain structure. However, little is known about HAP40 and thus the biological relevance of this structure. In this project, we transduced cells with fluorescently labelled recombinant apo-Htt or Htt-HAP40 to determine if HAP40 must be complexed with huntingtin in order for huntingtin to have biological activity. This method has not been implemented in HD research and may also improve current fluorescent microscopy models for huntingtin, as it has the advantage of looking at full-length protein rather than small fragments. We also found that with the huntingtin lowering drug branaplam, there is a linear correlation between huntingtin and HAP40 levels, where HAP40 levels will decrease when huntingtin levels are directly decreased as detected by western blot analysis. Furthermore, we found that this lowering effect by branaplam ameliorates DNA repair deficits in HD. With the potential for branaplam to become a treatment for HD, we should continue to test its effect on other HD-associated phenotypes to determine the effect of huntingtin and downstream HAP40 lowering. / Thesis / Master of Health Sciences (MSc)

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No description available.

The study of the aging process is the investigation as to how the passage of time affects cells, organs, and organisms. Aging is a very complex and incompletely understood phenomenon. This is reflected by the number of theories attributing aging to a variety of causative factors such as: (1) the somatic mutations occurring spontaneously or produced by ionizing radiation, which are thought to have some effect on again but are not responsible for the normal process; (2) the alteration of macromolecules as the cells of an organism age forming neoantigens and functioning in the autoimmune reactions; the Cross-linkage theory which maintains that large molecules necessary for life processes, such as deoxyribonucleic acid (DNA) and collagen are progressively immobilized in all cells and tissues by cross-linkage. Aging has also been studied in relation to the self-destructive “programmed death” characteristic of some parts of embryological development. Moreover, senescent changes involve different kinds of cells and tissues in the organism and therefore various mechanisms must occur. For example, the aging of postmitotic cells, such as neurons and cardiac cells probably proceeds by a different mechanism than the proliferating tissues, such as the skin, the gut lining, and the blood forming elements. It is apparent that there is probably no single aging process, but a series of aging processes which natural selection would tend to synchronize even if the causes were physiologically independent.

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This thesis successfully integrates laminate microfluidic devices with an analytic Surface Plasmon Resonance (SPR) instrument. Integration was accomplished at low-cost using materials such as polydimethylsiloxane (PDMS), Poly(methyl methacrylate) (PMMA), Tygon tubing, and a 3-way stopcock. The main components of this thesis are the design and fabrication of the low-cost, in-house fluidics that can integrate with upstream microfluidics and the validation of the in-house fluidics using the Biosensing Instruments BI-2000 SPR instrument. The low-cost fluidics was designed and fabricated “in-house” using a novel investment casting technique that required the use of laser cutting technology to make a master cast, and candle wax to make the fluidic flow gasket. Integration of upstream microfluidic devices is the next step towards fully integrated point-of-care (POC) diagnostics. Development of low-cost POC diagnostics will enable physicians to diagnosis patients outside of clinical settings, granting treatment access to a much wider population. Surface Plasmon Resonance is used for its detection abilities combined with its ability to perform real-time sample analysis. Validation of the in-house fluidics was accomplished by conducting (2) experiments: (1) to compare the angular shift elicited by ethanol solutions between in-house fluidics, factory fluidics, and the literature, and (2) to compare the angular shift between in-house fluidics and factory fluidics caused by the cleaving of fibroblasts from the SPR sensor chip. Successful comparisons made in both experiments proved successful development of low-cost fluidics that could integrate upstream microfluidic devices.

Cutaneous lupus erythematosus (CLE) is an autoimmune disease that resolves with or without permanent scars depending on the subtype. Interferons (IFNs), including the skin specific IFNλ mainly activate STAT1, which results in inflammation in CLE and may play a significant role in scar formation in chronic discoid CLE. IL-22 activates STAT3 and it is emerging as a mediator with significant impact on normal wound repair, epidermal hyperproliferation and prevention of fibrosis. This work focussed on understanding the regulation and functional impact of IL-22 and IFNλ on skin cells. The counter-regulatory effect of IL-22 on the activities of IFNλ was assessed through downstream interferon stimulated genes (ISGs) expression in healthy and CLE keratinocytes. Cell proliferation and gap closure were investigated in skin resident cells using cell trace dye and scratch assay. Dermal fibroblasts were assessed for the presence of IFNλR1 and IL-22R1, downstream activities of the receptors. Results showed that IL-22 accelerated “scratch” closure in keratinocytes while IFNλ caused a delay in closure. IL-22 significantly downregulated IFNλ-induced chemokines expression in healthy, but not CLE keratinocytes. Reduced IL-22R1 expression and “STAT3 signature genes” was observed in CLE keratinocytes. A key finding of this project is that dermal fibroblasts respond to both IFNλ and IL-22. This work shows that IL-22 can reduce the damaging effect of IFNs in inflamed skin and also identifies dermal fibroblasts as important cells in skin immune responses. In conclusion, IL-10 family members can have both beneficial and destructive effects on the skin organ depending on the micro milieu and cell-type involved. Manipulating the balance of IL-10 family members in the skin may offer new therapeutic approach for both psoriasis and CLE. / University of Bradford and Centre for Skin Sciences

The thesis presents a rational path for the optimization of the selection of optical treatment parameters in photobiomodulation of human skin fibroblasts. The project begins with an extensive analysis of 90 bibliographic reports in photobiomodulation published between 1985 and 2015, and revealed major inconsistencies in optical parameters selected for clinical applications. Seeking greater clarity for optimal parameter choice, a systematic approach to disentangle the multiple factors underpinning the response of human dermal fibroblasts in vitro to visible and near-infra red (NIR) light was employed. Light-based devices were constructed to specifically and systematically screen the optical parameter window (i.e. wavelength, irradiance and dose) observed in literature. Additionally, critical culture and treatment conditions that have dramatic impact on the outcome of specific light treatment of these human skin dermal cells were identified. In particular, environmental oxygen concentration, cell confluency and serum concentration were all found to have a great effect on the response of dermal fibroblasts to light. In parallel, the induction of reactive oxygen species (ROS) by short visible wavelengths on two dermal fibroblast sub-populations or lineage, reticular and papillary, was monitored by live-cell imaging. The ROS species were found to be created in or close to mitochondria. Lastly, gene expression studies revealed a strong impact of short visible wavelengths, as compared to long and NIR wavelengths on both subpopulations of human dermal fibroblasts. In particular, blue light (450 nm) specifically down-regulated proliferation, metabolism and protein synthesis molecular pathways. At the protein level, 450-nm light inhibited the production of procollagen I in human reticular and papillary fibroblasts in a dose-dependent manner. Gene expression results were in agreement i.e., the same light parameter down-regulated collagen fiber genes, integrins and up-regulated collagenase MMP1. This thesis concludes with a chapter presenting a characterization of the accuracy of a potential translation tool for the prediction of optical photon density inside human skin. / Marie Skłodowska-Curie Actions.