Biomaterials-tissue interaction of an injectable collagen-genipin gel in a rodent hemi-resection model of spinal cord injury

Thesis (Ph. D. in Medical Engineering and Medical Physics)--Harvard-MIT Program in Health Sciences and Technology, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Spinal cord injury (SCI) is a significant health issue resulting in life-long disability and associated secondary complications, affecting approximately 300,000 individuals in the United States. Primary barriers to functional recovery after SCI include the formation of a growth inhibitory astrocyte scar at the lesion border and a lack of a supportive stroma within the defect allowing for axon regeneration. Interestingly, in animals capable of spinal cord regeneration, astrocytes create a tissue bridge across the injury site to facilitate the regeneration of axons through the defect and thus enable functional recovery. The overall goal of this thesis was to develop an injectable collagen-genipin (Col-Gen) hydrogel to facilitate the intrinsic regenerative response after SCI by promoting the population of the defect with astrocytes through a provisional scaffold pennissive of astrocyte migration. The specific aims of the thesis involved: 1) development and materials characterization of an injectable collagen hydrogel for neural tissue regeneration, capable of undergoing covalent crosslinking in vivo; 2) evaluation of the permissiveness of Col-Gen gels with and without Fibroblast growth factor-2 (FGF-2), a known astrocyte chemoattractant, incorporated within lipid microtubules (LMTs) to infiltration by primary astrocytes using an in vitro cellular outgrowth assay; 3) evaluation of select formulations of the gel, based on the in vitro findings, in a standardized hemi-resection defect in the rat spinal cord. Functional, locomotor, and histopathological outcome measures, recorded up to 4 weeks post-SCI were correlated with each other and with micro MRI studies. In vivo, the implantation of Col-Gen gels containing FGF-2 LMTs resulted in the enhancement of astrocyte, blood vessel, and laminin infiltration of the defect; increased the amount of spinal cord tissue spared from secondary degeneration; and increased functional recovery, at four weeks post injury as compared to control or Col-Gen treatment groups. Micro MRI was found to be a suitable modality to nondestructively observe the features of the injury in situ. This work commends an injectable, covalently cross-linkable formulation of collagen gel incorporating FGF-2-releasing LMTs for further investigation for the treatment of SCI.by Daniel J. Macaya.Ph.D.in Medical Engineering and Medical Physic

All-plastic electrochemical transistor for glucose sensing using a ferrocene mediator.

We demonstrate a glucose sensor based on an organic electrochemical transistor (OECT) in which the channel, source, drain, and gate electrodes are made from the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS). The OECT employs a ferrocene mediator to shuttle electrons between the enzyme glucose oxidase and a PEDOT:PSS gate electrode. The device can be fabricated using a one-layer patterning process and offers glucose detection down to the micromolar range, consistent with levels present in human saliva

Zero-order controlled release of ciprofloxacin-HCl from a reservoir-based, bioresorbable and elastomeric device

A reservoir-based device constructed of a completely biodegradable elastomer can enable several new implantation and insertion options for localized drug therapy, particularly in the case of urological therapies. We performed an in vitro performance evaluation of an implantable, bio-resorbable device that supplies short-term controlled release of ciprofloxacin-HCl (CIP). The proposed device functions through a combination of osmosis and diffusion mechanisms to release CIP for short-term therapies of a few weeks duration. Poly(glycerol-co-sebacic acid) (PGS) was cast in a tubular geometry with solid drug powder packed into its core and a micro-machined release orifice drilled through its wall. Drug release experiments were performed to determine the effective release rate from a single orifice and the range of orifice sizes in which controlled zero-order release was the main form of drug expulsion from the device. It is demonstrated that PGS is sufficiently permeable to water to allow the design of an elementary osmotic pump for drug delivery. Indeed, PGS's water permeability is several orders of magnitude larger than commonly used cellulose acetate for elementary osmotic pumps.Deshpande Center for Technological InnovationSamsung Scholarship Foundatio

Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant’s sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes use of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. The resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for complex phenotypes, such as recalcitrance

Management Pearls on the Treatment of Actinic Keratoses and Field Cancerization

Field cancerization (FC) is a chronic disease involving multiple clinical and subclinical actinic keratoses (AK) on large photo-exposed surfaces with multifocal areas of dysplasia and precancerous changes. Patients and treatment must be properly monitored and managed to avoid aggravation and progression of the disease. Management of actinic keratoses includes lesion-directed treatments, such as cryotherapy and field-directed therapies. Field-directed therapies may have the potential to address subclinical damage, reduce AK recurrence rates and potentially reduce the risk of squamous cell carcinoma development. Multiple studies have demonstrated the efficacy of field-directed treatments, including 5-fluorouracil, photodynamic therapy, imiquimod, chemical exfoliation with trichloroacetic acid and diclofenac gel, for multiple AK and FC. The choice of therapy should be based on multiple factors, such as efficacy, tolerability, patient risk profile, costs and cosmetic results. Management of AK includes not only treatment but also prevention. Medical devices, such as sunscreens containing liposome-encapsulated DNA repair enzymes, can repair DNA damage associated with chronic UV radiation and reduce the number of new AK lesions. Here we provide therapeutic pearls and expert opinions on the treatment of AK and FC (as monotherapy or in combination) with the overall aim to achieve better, faster, and well-tolerated clinical responses

Isotopic Characterization of Water Masses in the Southeast Pacific Region: Paleoceanographic Implications

In this study, we used stable isotopes of oxygen (δ18O), deuterium (δD), and dissolved inorganic carbon (δ13CDIC) in combination with temperature, salinity, oxygen, and nutrient concentrations to characterize the coastal (71°–78°W) and an oceanic (82°–98°W) water masses (SAAW—Subantarctic Surface Water; STW—Subtropical Water; ESSW—Equatorial Subsurface water; AAIW—Antarctic Intermediate Water; PDW—Pacific Deep Water) of the Southeast Pacific (SEP). The results show that δ18O and δD can be used to differentiate between SAAW-STW, SAAW-ESSW, and ESSW-AAIW. δ13CDIC signatures can be used to differentiate between STW-ESSW (oceanic section), SAAW-ESSW, ESSW-AAIW, and AAIW-PDW. Compared with the oceanic section, our new coastal section highlights differences in both the chemistry and geometry of water masses above 1,000 m. Previous paleoceanographic studies using marine sediments from the SEP continental margin used the present-day hydrological oceanic transect to compare against, as the coastal section was not sufficiently characterized. We suggest that our new results of the coastal section should be used for past characterizations of the SEP water masses that are usually based on continental margin sediment samples

Finding New Cell Wall Regulatory Genes in Populus trichocarpa Using Multiple Lines of Evidence.

Understanding the regulatory network controlling cell wall biosynthesis is of great interest in Populus trichocarpa, both because of its status as a model woody perennial and its importance for lignocellulosic products. We searched for genes with putatively unknown roles in regulating cell wall biosynthesis using an extended network-based Lines of Evidence (LOE) pipeline to combine multiple omics data sets in P. trichocarpa, including gene coexpression, gene comethylation, population level pairwise SNP correlations, and two distinct SNP-metabolite Genome Wide Association Study (GWAS) layers. By incorporating validation, ranking, and filtering approaches we produced a list of nine high priority gene candidates for involvement in the regulation of cell wall biosynthesis. We subsequently performed a detailed investigation of candidate gene GROWTH-REGULATING FACTOR 9 (PtGRF9). To investigate the role of PtGRF9 in regulating cell wall biosynthesis, we assessed the genome-wide connections of PtGRF9 and a paralog across data layers with functional enrichment analyses, predictive transcription factor binding site analysis, and an independent comparison to eQTN data. Our findings indicate that PtGRF9 likely affects the cell wall by directly repressing genes involved in cell wall biosynthesis, such as PtCCoAOMT and PtMYB.41, and indirectly by regulating homeobox genes. Furthermore, evidence suggests that PtGRF9 paralogs may act as transcriptional co-regulators that direct the global energy usage of the plant. Using our extended pipeline, we show multiple lines of evidence implicating the involvement of these genes in cell wall regulatory functions and demonstrate the value of this method for prioritizing candidate genes for experimental validation

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic and functional studies

Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing

Finding New Cell Wall Regulatory Genes in Populus trichocarpa Using Multiple Lines of Evidence

Understanding the regulatory network controlling cell wall biosynthesis is of great interest in Populus trichocarpa, both because of its status as a model woody perennial and its importance for lignocellulosic products. We searched for genes with putatively unknown roles in regulating cell wall biosynthesis using an extended network-based Lines of Evidence (LOE) pipeline to combine multiple omics data sets in P. trichocarpa, including gene coexpression, gene comethylation, population level pairwise SNP correlations, and two distinct SNP-metabolite Genome Wide Association Study (GWAS) layers. By incorporating validation, ranking, and filtering approaches we produced a list of nine high priority gene candidates for involvement in the regulation of cell wall biosynthesis. We subsequently performed a detailed investigation of candidate gene GROWTH-REGULATING FACTOR 9 (PtGRF9). To investigate the role of PtGRF9 in regulating cell wall biosynthesis, we assessed the genome-wide connections of PtGRF9 and a paralog across data layers with functional enrichment analyses, predictive transcription factor binding site analysis, and an independent comparison to eQTN data. Our findings indicate that PtGRF9 likely affects the cell wall by directly repressing genes involved in cell wall biosynthesis, such as PtCCoAOMT and PtMYB.41, and indirectly by regulating homeobox genes. Furthermore, evidence suggests that PtGRF9 paralogs may act as transcriptional co-regulators that direct the global energy usage of the plant. Using our extended pipeline, we show multiple lines of evidence implicating the involvement of these genes in cell wall regulatory functions and demonstrate the value of this method for prioritizing candidate genes for experimental validation

Phosphomannomutase deficiency (PMM2-CDG): Ataxia and cerebellar assessment

Background: Phosphomannomutase deficiency (PMM2-CDG) is the most frequent congenital disorder of glycosylation. The cerebellum is nearly always affected in PMM2-CDG patients, a cerebellar atrophy progression is observed, and cerebellar dysfunction is their main daily functional limitation. Different therapeutic agents are under development, and clinical evaluation of drug candidates will require a standardized score of cerebellar dysfunction. We aim to assess the validity of the International Cooperative Ataxia Rating Scale (ICARS) in children and adolescents with genetically confirmed PMM2-CDG deficiency. We compare ICARS results with the Nijmegen Pediatric CDG Rating Scale (NPCRS), neuroimaging, intelligence quotient (IQ) and molecular data. Methods: Our observational study included 13 PMM2-CDG patients and 21 control subjects. Ethical permissions and informed consents were obtained. Three independent child neurologists rated PMM2-CDG patients and control subjects using the ICARS. A single clinician administered the NPCRS. All patients underwent brain MRI, and the relative diameter of the midsagittal vermis was measured. Psychometric evaluations were available in six patients. The Mann-Whitney U test was used to compare ICARS between patients and controls. To evaluate inter-observer agreement in patients' ICARS ratings, intraclass correlation coefficients (ICC) were calculated. ICARS internal consistency was evaluated using Cronbach's alpha. Spearman's rank correlation coefficient test was used to correlate ICARS with NPCRS, midsagittal vermis relative diameter and IQ. Results: ICARS and ICARS subscores differed between patients and controls (p < 0.001). Interobserver agreement of ICARS was "almost perfect" (ICC = 0.99), with a "good" internal reliability (Cronbach's alpha = 0.72). ICARS was significantly correlated with the total NPCRS score (rs 0.90, p < 0.001). However, there was no agreement regarding categories of severity. Regarding neuroimaging, inverse correlations between ICARS and midsagittal vermis relative diameter (rs -0.85, p = 0.003) and IQ (rs -0.94, p = 0.005) were found. Patients bearing p.E93A, p.C241S or p.R162W mutations presented a milder phenotype. Conclusions: ICARS is a reliable instrument for assessment of PMM2-CDG patients, without significant inter-rater variability. Despite our limited sample size, the results show a good correlation between functional cerebellar assessment, IQ and neuroimagingFor the first a correlation between ICARS, neuroimaging and IQ in PMM2-CDG patients has been demonstratedThe work was supported by national grants PI14/00021, PI11/01096, PI11/01250, and PI10/00455 from the National Plan on I+D+I, cofinanced by ISC-III (Subdirección General de Evaluación y Fomento de la Investigación Sanitaria) and FEDER (Fondo Europeo de Desarrollo Regional) and IPT-2012- 0561-010000 from MINECO. Three research groups (U-746, U-737 and U703) from the Centre for Biomedical Research on Rare Diseases (CIBER-ER), Instituto de Salud Carlos III, Spain, have worked together for the present stud