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Pitx3Null Mutant (Striatal Dopamine-Deficient) Mice Have Exaggerated Spiny Projection Neuron Responses to l-DOPA and D1 Agonism and Lack Baseline Striatonigral Spiking
Get PDFL-3,4 dihidroxyphenylalanine (l-DOPA) strongly stimulates motor activity in parkinsonian patients and animal models of Parkinson\u27s disease. Severe striatal dopamine (DA) loss characterizes Parkinson\u27s disease and its animal models. Given the canonical rate model of Parkinson\u27s Disease pathophysiology based on differences in DA pharmacology manifesting as electrophysiological differences in striatal projection neuron (SPN) spike rates, SPNs should increase spiking during the motor response to l-DOPA. In fact, stimulating specific subsets of these neurons to spike in freely-moving wild type and parkinsonian animals causes or inhibits motor activity as predicted. However, pharmacological effects of DA deficiency, let alone those of DA replacement, on SPN spiking activity in freely-moving animals are poorly studied and ultimately unknown. Showing the activity of SPNs of both in-/direct pathways may help elucidate mechanisms by which l-DOPA increases motor activity to normal and sometimes abnormal levels; such mechanistic information would advance understanding about how DA is such a potent motor stimulant.
To this end, I devised a Top-hat u-array (with microdrive) for recording in the striatum while stimulating the reticulated substantia nigra. Using my micro-array, I tested l-DOPA\u27s acute effect on SPN spiking activity within contexts that varied in DA deficiency according to the Pitx3Null mouse\u27s Parkinson\u27s-like gradient of striatal DA denervation. Evidently, chronic DA denervation renders SPNs hyper-responsive to l-DOPA and a D1 agonist, SKF 81297, as indicated by exaggerated SPN spike rate responses biased by low baselines in Pitx3Null mice compared to wild-type mice. However, this may be a motor network effect on spiking as it was found in both dorsal (DA-denervated) and non-dorsal (having residual DA) Pitx3Null striatal regions. Furthermore, antidromically identifying dorsal SPNs allowed us to putatively distinguish a particularly relevant subset (striatonigral, D1-SPNs or d[irect]SPNs) known to elicit movement; serendipitously we also identified putative fibers of passage that strongly resembled striatal interneurons. D1-SPNs in Pitx3Null animals had baselines about an order of magnitude significantly below those in wild-type, and all increased firing more so in Pitx3Null than wild-type mice after drug injections, which lends some credence to the hypothesis that direct pathway SPNs are hyper-responsive during l-DOPA-induced normal and abnormal motor behavior secondary to DA depletion. Furthermore, they uncover a need to incorporate more neural factors in explaining electrophysiological effects attributable to DA denervation and restoration pharmacology. The latter data (putative fibers) tempt the interpretation that cortical axons of passage are being mistaken for striatal fast-spiking interneurons in the literature more often than not
Back of the Envelope Calculations Regarding Alexandrov, et al. 2009
Get PDFThe paper entitled “DNA Breathing Dynamics in the Presence of a Terahertz Field” written by Alexandrov, Gelev, Bishop, Usheva, and Rasmussen that was posted on the Physics ArXiv on October 28, 2009, dealt with the effects of terahertz electromagnetic radiation on the DNA molecule. Figure 4 of the paper indicates that a segment of DNA that is between 60 and 70 base pairs in length will develop a “bubble” (i.e. separation of the hydrogen bonds between base pairs) at a location of about 45 base pairs, when exposed to 2 THz electromagnetic radiation for a time period of at least 40 picoseconds. Based upon assumptions regarding the model of biological quantum logic in DNA, some rapid calculations might provide insight into why such a specific change in the conformation of DNA occurs
Discovery of Natural Product-based Antimycobacterial Agents Effective against Non-replicating Bacilli
Get PDFNew antimycobacterial molecules that kill non-replicating Mycobacterium tuberculosis (Mtb) were identified by screening libraries of synthetic natural products. De novo screening of a 400-membered library of aurachin RE analogs resulted in discovery of UT-317 ((R)-20). UT-317 is a selective vitamin K2 biosynthesis (MenA) inhibitor that killed replicating and non-replicating Mtb at 2.31 μg/mL (MIC) and 0.85 μg/mL, respectively. A 50-membered library of capuramycin analogs was evaluated in their enzymatic inhibitory activities against translocase I (MraY/MurX) and prenyl-phosphate-GlcNAc-1-phosphate transferase (WecA). UT-01320 (45) is identified as a selective WecA inhibitor that kills both replicating and non-replicating Mtb at 1.50 μg/mL (MIC) and 2.58 μg/mL, respectively. UT-01320 killed the intracellular Mtb much faster than the first-line TB drugs such as isoniazid and rifampicin. A strong antimycobacterial agent, UT-800 (64) was identified by screening of a 50-membered library of pleuromutilin derivatives. UT-800 is a protein biosynthesis (50s ribosome) inhibitor which has activity focused against Mtb. UT-800 killed replicating and non-replicating Mtb at 0.83 μg/mL (MIC) and 1.20 μg/mL, respectively. In the course of these works, fluorescent probes, Park’s nucleotide-Nε-C6-FITC (32, for MraY/MurX) and UDP-glucosamine-C6-FITC (46, for WecA) were developed. These fluorescent probes enable us to screen the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase enzyme superfamily (e.g. MraY/MurX, WecA, AlgH, and DPAGT1) in high-throughput manner
Revealing a Non-canonical Role of Anti-apoptotic MCL-1 in Early Embryonic Development
Get PDFMCL-1, a well-known pro-survival BCL-2 family member, is indispensable for the survival of various cellular lineages and is also among the most frequently amplified genes in a variety of human malignancies. Gene ablation studies previously revealed that Mcl-1 deficiency leads to embryonic lethality around E3.5 during peri-implantation stage. Strikingly, the study did not detect any increase in apoptotic cells of the blastocyst, indicating a function of MCL-1 beyond regulating apoptosis. Our previous studies revealed an unrecognized role of MCL-1 in promoting mitochondrial physiology, which is independent of its classical anti-apoptotic function and requires being imported into the mitochondrial matrix. In order to understand the role of MCL-1 in early embryonic development, we used CRISPR-Cas9 to target Mcl-1’s start codon on established embryonic stem cells (ESCs). This approach resulted in the establishment of ESCs in which MCL-1’s N-terminus was truncated. Biochemical evaluation revealed that Nterminal- deleted MCL-1 retains anti-apoptotic function. However, this truncated MCL-1 is restricted to the mitochondrial outer membrane and functionally these mutated ESCs showed a dramatic defect in differentiation into the three embryonic germ layers- ectoderm, mesoderm, and ectoderm. These data suggest that in addition to MCL-1’s required antagonism of cell death by the C-terminal region, MCL-1’s N-terminus is required for efficient cellular differentiation, potentially by facilitating MCL-1’s import into the mitochondrial matrix
The Pathway to the use of a Structured and Supported Approach to Debriefing
Get PDFDebriefing plays a critical role in the transfer of knowledge in simulation education. Various methods are used in the debriefing process based on a variety of factors; this presentation sheds light on the pathway to the use of a structured and supported approach as traversed in the Department of Occupational Therapy at the University of Tennessee Healthcare Center as well as reflections and lessons learned along the way. Engagement in this session will allow learners to further develop their debriefing skills through exposure to information about strategies to use in the debriefing process that can be used as part of simulation learning experiences to foster reflective thinking and facilitate transformative learning. Through listening to this account and the related research presented, learners will be able to list 5 steps involved in a holistic debriefing process, identify 3 ways to address the emotional charge resulting from SIM encounters, and discuss specific strategies to use to support students in connecting new learning to a larger clinical environment. Research supporting the use of the structured and supported model for debriefing will be shared along with observations of student and faculty responses that have come as a result of the implementation of these techniques in the experience of the presenters. Challenges in debriefing as well as plans for continued growth in this area will be discussed as part of this session as well
Perceptions of EMR Documentation in the Home Setting
Get PDFThe use of technology in healthcare is ever increasing in our hospitals, doctor’s offices, and clinics. Studies have been done to ascertain the perceptions of patients on the use of technology in the places that provide them care. This project was done for a population that does not always receive care where the clinician is. The care is in their home by clinicians who are using laptop computers and documenting in the EMR. Home care and hospice patients or their caregivers were asked to complete a survey with seven questions scored with a 5-point Likert scale to find that they feel positive that the computer contributes to better care and helps check for errors, the clinicians are listening, and does not affect their relationship with the clinician. The biggest concern of this population is the safety and security of their information
Synthesis of 20S-Hydroxyvitamin D3 Analogs and Their 1α-Hydroxyl Derivatives as Potent Anti-inflammatory Agents
Get PDFRheumatoid arthritis (RA) is one of the autoimmune diseases, and is affecting 2.5 million Americans in total. Among the treatment options of RA, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the only steroidal drug used clinically for anti-inflammatory and immune diseases. However, long-term use of 1,25(OH)2D3 (625 µg/day) in human would result in hypercalcemia (toxicity), and 1,25(OH)2D3 has substantial hypercalcemic effects (toxicity) in mice at a dose as low as only 2 µg/kg. Fortunately, during the investigation of novel metabolic pathway of vitamin D3 by cytochrome P450 enzymes, we found 20S-hydroxyvitamin D3 [20S(OH)D3] as a good lead compound. 20S(OH)D3 suppressed disease symptoms at 2 µg/kg in collagen-induced arthritis model, and high doses of 20S(OH)D3 (up to 30 µg/kg) do not cause hypercalcemia in rats or mice. Thus 20S(OH)D3 has the potential to be structurally optimized for providing anti-inflammatory agents without toxicity. In this study, four series of 20S(OH)D3 analogs have been synthesized and studied, they are C20 Gemini analogs, C24-hydroxlated analogs, C23-hydroxlated analogs and C24 modified analogs together with their 1α-hydroxylated derivatives. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20S(OH)D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR. Bioactive vitamin D3 metabolites 20S,24S-dihydroxyvitamin D3 [20S,24S(OH)2D3] and 20S,24R-dihydroxyvitamin D3 [20S,24R(OH)2D3] were chemically synthesized and confirmed to be identical to their enzymatically generated counterparts. The absolute configurations at C24 and its influence on the kinetics of 1α-hydroxylation by CYP27B1 were determined. Their corresponding 1α-hydroxyl derivatives were subsequently produced. Biological comparisons of these products showed different properties with respect to vitamin D3 receptor activation, anti-inflammatory activity, and anti-proliferative activity, with 1α,20S,24R(OH)2D3 being the most potent compound. The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time, and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory and anti-proliferative activities. Four C24 modified analogs of 20S(OH)D3 were chemically synthesized and comprehensively tested against different activities together with their 1α-hydroxyl derivatives. Metabolism of 20S(OH)D3 analogs against cytochrome P450 27B1 (CYP27B1, activation enzyme) and CYP24A1 (catabolism enzyme) suggested that they are better substrates of both enzymes than 20S(OH)D3, and can be activated (1α-hydroxylated) by CYP27B1 except 23-amide which is not a substrate but an inhibitor of CYP27B1. Their 1α-OH derivatives were potent vitamin D receptor (VDR) agonists comparable with 1,25(OH)2D3 although they themselves showed weak or none VDR stimulation activity in three cell lines. To understand the molecular interactions between these analog and VDR, two analogs together with 20S(OH)D3 and 1,25(OH)2D3 were co-crystalized with human VDR. These analogs and 1α-OH derivatives significantly upregulated the mRNA expression of VDR target genes, suggesting their actions via VDR, at least partially. In addition, their anti-inflammatory activities have been investigated in aspect of IFNγ inhibition in splenocytes. This study demonstrates the mechanisms of action of 20S(OH)D3 anlogs, is of great importance for future drug development of anti-inflammatory agents. From the above-mentioned studies, we learned that the introduction of 1α-hydroxy could potentiate the anti-inflammatory activities of 20S(OH)D3 and its anlogs. Thus it would be beneficial to further investigate the 1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3] analogs. 1,20S(OH)2D3 was chemically synthesized for the first time. A semi-reduced intermediate of the Birch reduction for 1α-OH formation was obtained for the first time, and thus was used to propose the reaction mechanism. X-ray crystallography analysis of the key intermediate confirmed the formation of 1α-OH. 1,20S(OH)2D3 binds efficiently in vitamin D receptor (VDR), being similar with its native ligand 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, their co-crystal structures revealed differential molecular interactions of 20S-OH and 25-OH in VDR, which may help understand their biological activities. In addition, 1,20S(OH)2D3 functions as a VDR agonist with stronger/comparable activities than/with 1,25(OH)2D3 in aspects of VDR stimulation and regulating VDR downstream genes, and inhibition of inflammatory markers. This study offers a convenient synthetic route using a novel intermediate 1α,3β-diacetoxypregn-5-en-20-one, and provides molecular basis of design for drug development of 1,20S(OH)2D3 and its analogs. Overall, we have synthesized and biologically evaluated four series of 20S(OH)D3 analogs for their potential applications in anti-inflammatory diseases such as RA. The synthetic scheme of 1,20S(OH)2D3 could pioneer future development of its analogs. These findings will provide important guidance for the development of next generation anti-RA agents using 20S(OH)2D3 scaffold
Biophysical and Biochemical Screening Approaches for Antimicrobial Drug Discovery Targeting S. aureus ClpP
Get PDFThe discovery of antibacterial drugs has been among most significant achievements of mankind in saving millions of lives across the planet from infectious diseases. With rise in resistance to almost all existing chemotypes, the design of next generation novel antibiotics has become much more challenging and difficult. The early 21st century witnessed the advancement of multiple novel chemotypes during golden age of antibiotics however the pace of antibiotic drug discovery has slowed down tremendously, contributing to life threatening antimicrobial discovery void since 1980’s. Therefore the need to develop novel antibiotics with unique mechanism of action to leverage against multi drug resistance pathogens, is paramount. In this direction the Caseinolytic Protease P (ClpP) is an emerging drug discovery target with significant potential for treatment of recalcitrant biofilm forming infections from pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) This dissertation highlights the ongoing efforts to facilitate the discovery of novel non peptidic ClpP activator compounds and improvement of pharmacological profile of existing ClpP targeting Acyldepsipeptides (ADEPs) series antibiotics. The chapter one discusses the history and synopsis of conventional antibiotics drug discovery screening approaches, and transitions to modern era structure or fragment based screening approaches. The merits and challenges of such approaches of targeting a well conserved bacterial protease (ClpP) are discussed along with dissertation aims toward development of biophysical and biochemical screening approaches. Chapter two discusses optimization of thermal shift assay as primary screening assay for ClpP and its utility toward screening of fragment collections and buffer conditions. Chapter three discussed the development of a site specific Fluorescence Polarization based FP probe based on ADEP scaffold and its utility as a robust high throughput capable primary screening assay for screening of diverse collections ranging from bioactives to fragments. Chapter four discusses development of a label free Surface Plasmon Resonance (SPR) based assay geared toward screening of fragment as well as in house small and large (ADEP analogs) series compounds in addition to determining full kinetics for lead prioritization. Chapter five discusses the results of multiple screening campaigns utilizing combination of above assays to generate multiple hits with superior ligand efficiency and chemical tractability. Chapter six concludes with analysis of the best of compounds among individual series or from screening campaigns and highlights effectiveness of above screening assays toward hit exploration along with outlook on anticipated challenges and future directions
Translational Pharmacokinetic-Pharmacodynamic Modeling and Simulation in the Development of Spectinamides, a Novel Class of Anti-Tuberculosis Agents
Get PDFNew chemotherapeutic agents are urgently needed to control the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of tuberculosis, which still remains an important public health challenge globally. Recently, spectinamides have emerged as a novel class of anti-tuberculosis agents that overcomethe native drug efflux. Spectinamides bind to the 30S bacterial ribosomal subunit which interferes with ribosomal translocation, and ultimately results in inhibition of protein synthesis. They have potent in vitro activity against drug resistant Mycobacterium tuberculosis (Mtb), and also demonstrated sustained efficacy in (Mtb)-infected mouse models. Pharmacokinetic (PK)/ pharmacodynamic (PD) analyses play a critical role in identifying the optimum dosing regimen for new treatments. In this dissertation, I hypothesized that the application of translational PK/PD modeling and simulation techniques would facilitate rational dosage regimen design for spectinamides.
To characterize the dose-exposure-response of Lee 1810, a dose-fractionation study was performed in BALB/c mice infected with a low dose aerosol of (Mtb). Dosing with different dosing regimens was continued for 4 weeks with two blood samples obtained from each mice in the last week, followed by a washout period after which the mice were sacrificed and the lungs removed for measurement of colony forming units (CFU). Drug concentrations in plasma were analyzed with a validated LC-MS/MS method followed by a population PK analysis which also included as anchor point the data of a PK study in healthy mice with intensive sampling. A model for natural bacterial growth in Mtb infection in untreated mice was built from data on the natural history of Mtb infection in mice obtained from previously performed studies and from the literature. Based on the individual post hoc estimates from the population PK modeling, a sequential PK/PD analysis was performed by linking the PK model with the bacterial growth model via an exposure-dependent bacterial kill function that included a sigmoid Emax model for describing the overall rate of change in lung CFU with different dosing regimens. A two-compartment model with first-order absorption was used to describe the pharmacokinetics of Lee 1810. The average absorption rate constant (Ka), clearance (CL/F), volume of the central compartment (Vc/F), intercompartmental clearance (Q/F), and volume of the peripheral compartment (Vp/F) was estimated to be 2.31 h-1,1.17 L/h/kg, 0.435 L/kg, 0.0191 L/h/kg, and 0.161 L/kg, respectively. The inter-individual variability in CL/F was estimated as 19.9 %. The pharmacokinetics of Lee 1810 was found to be different between healthy and infected mice with the later having 56.5% lower CL/F, 69% lower Vc/F and 69.6% lower Q/F. The two-subpopulation model could successfully describe the natural bacterial growth. The replication rate constant (Krep) of Mtb was calculated as 0.0327 h-1 which is consistent with values reported in the literature. The death rate constant induced by the immune system (Kir) was 0.00303 h-1, cell countof fast growing population at the initiation of the infection (N1,0) was 1.93 Log CFU and maximum number of bacteria (Nmax) was 6.44 Log CFU. The inter-individual variability in Krep and Nmax was estimated as 70.8 % and 54.7%, respectively. The bacterial kill induced by the drug was described using a sigmoid Emax model. The drug effect parameters (EC50), maximum kill rate (Emax) and Hill coefficient (y), were estimated as 239 μg/mL, 11.9 h-1 and 2.40 respectively. A Hill coefficient substantially greater than 1 is a typical characteristic of concentration-dependent killing. The concentration dependent killing characteristic of Lee 1810 supports its intermittent dosing.
Poor permeability of spectinamides across the gut limits its oral use. Additionally, since the lungs are the main site of infection in pulmonary TB, the efficacy of lead spectinamide Lee 1599 was evaluated after intratracheal (IT) administration in a mouse model of Mtb infection. A dose of 200 mg/kg TIW (3 days a week) for 28 days resulted in excellent efficacy with 2.2 Log CFU reduction in the lungs. Based on these observations, a comparative biodistribution study of Lee 1599 was performed after IT and SC administration in mice. Plasma and tissue samples were collected at pre-specified time points. The drug was extracted from plasma and homogenized tissues after protein precipitation and analyzed with an LC-MS/MS assay. The rate and extent of absorption was almost two times higher with IT as compared to SC administration. As expected, the highest exposure of Lee 1599 after IT administration was attained in the lungs, which was 2.5 times higher than in plasma. This is highly desirable as lungs are the main site of infection in pulmonary tuberculosis. Overall, this study supports the pulmonary route as a potential pathway for the treatment of tuberculosis with Lee 1599.
Physiologically-based pharmacokinetic (PBPK) modeling and simulation is a powerful methodology used in support of dose selection for first-in-human studies. The objective was to develop a PBPK model for describing pharmacokinetics of Lee 1599 in rats and mice, and to extrapolate this PK behavior to humans. 10 mg/kg of Lee 1599 was administered intravenously to rats and 200 mg/kg subcutaneously to mice. The PBPK model was developed based on the observed rat plasma concentrations, physicochemical properties of Lee 1599, and in vitro data from its metabolism, protein binding and permeability. The concentration-time profile of Lee 1599 in rats was well described by the optimized PBPK model. The model was prospectively qualified by PBPK scaling from rats to mice and comparing predicted murine concentration-time profiles to observed plasma concentrations. This model was also successful in predicting murine PK with observed PK parameters within two-folds of predicted values. The model predicted, weight normalized human clearance of 0.25 L/h/kg was as expected less than the values in rats (0.666 L/h/kg) and mice (1.25 L/h/kg). The PBPK model predicted, a dose of 7.5 mg/kg and 27.5 mg/kg administered once daily via intravenous administration will be required to attain similar exposure as observed in mice after subcutaneous administration of 50 mg/kg and 200 mg/kg respectively. This model suggests that an efficacious systemic exposure can be achieved with daily doses feasible in humans, and may be useful during drug development for understanding the dose requirements for future human studies.
In conclusion, translational PK/PD approaches have been successfully used for the further development and characterization of spectinamides leads Lee 1599 and Lee 1810. The results from the above studies will be helpful in identifying and optimizing the dosing regimens which can strike a balance between bacterial reduction, adverse effects, and emergence of resistance
Investigating the Role of Dispatched in Hedgehog Ligand Transport and Delivery
Get PDFDuring the development of all metazoans, the Hedgehog (Hh) signaling pathway provides instructional cues influencing a variety of cellular processes. The pathway ligand, Hh, is dually lipidated by cholesterol and palmitate, which effectively anchors the molecule to the lipid bilayer of the signal producing cell. To complicate the Hh pathway induction process, the Hh ligand is often produced at a significant distance from the cells it influences. Only one known conserved molecule, Dispatched (Disp), can alleviate the membrane tethering imparted by Hh lipidation. Underscoring the importance of Disp protein during development, knockout animals succumb to lethality at E9.5, an exact phenocopy of the knockout of the essential signal transducer of the pathway: Smoothened. Furthermore, mutations within Disp have been found in patients with Holoprosencephaly, the most common cause of human forebrain malformations, which is frequently caused by inhibition of Hh signaling during development. Very little is known regarding the functional or regulatory mechanisms enabling Disp to transport and release Hh ligand. This dissertation aimed to narrow this gap and began with investigation into the role of Disp in a largely ignored mechanism of Hh ligand transport known as cytoneme-mediated morphogen transport. This method of morphogen transport utilizes fragile, thin cytoplasmic extensions which deliver cargo directly from the source of production to ligand responding cells. Through the use of a modified electron microscopy fixative, which we named MEM-fix, to maintain cytonemes for traditional cell biological analysis, I established an in vitro cytoneme system capable of modeling in vivo cytoneme biology. This in vitro cytoneme system uses Schneider 2 cells, an embryonically derived Drosphila cell line, which demonstrate competency to produce and utilize cytonemes as a mechanism of Hh transport. Equipped with this tool, I investigated the relationship between cytonemes and Hh pathway components. In doing so, I uncovered a previously unknown Disp requirement in cytoneme-mediated transport of Hh ligand and subsequently a Disp-mediated cytoneme stabilizing effect. Through these studies into Disp-mediated cytoneme transport, I identified a Disp cleavage event of Disp that regulates the ability to release Hh ligand. The second part of this dissertation details a collaborative effort in which we discovered that the Furin family of proprotein convertases facilitate the cleavage of Disp at a conserved site of the first extracellular loop. The Furin family has been implicated in cleaving ligands and receptors of other developmental signaling pathways, but no reports exist linking the Furin family to regulation of the Hh pathway. Therefore, we investigated the functional consequence of this Disp cleavage event, and our results suggest that this cleavage likely influences the proper trafficking of Hh for efficient release both in vitro and in vivo. To our knowledge, this is the first report of a regulatory protein partner controlling the activity of Disp in releasing Hh