Liquid Chromatography-Mass Spectrometry Strategies for in vivo Neurochemical Monitoring with Microdialysis.

Liquid chromatography mass spectrometry (LC-MS) is a powerful analytical tool for multi-analyte quantification. This method can be combined with microdialysis sampling to study small molecules and neuropeptides within discrete brain regions. This thesis focuses on the development of targeted LC-MS assays to analyze dialysate samples collected from awake animals to correlate neurochemical dynamics with behavior. Previous LC-MS assays used benzoyl chloride derivatization to enable quantification of 17 neurotransmitters and metabolites in dialysate samples. In this work, derivatization conditions were modified to improve sensitivity up to 25-fold and reduce complexity of the procedure. The assay was also expanded to 70 compounds including amino acids, polyamines (e.g., putrescine, spermidine, spermine), compounds from catecholamine biosynthesis pathways (e.g., tyrosine and tryptophan metabolic pathways), and trace amines (e.g., tyramine, octopamine, synephrine). Besides measurements in dialysate, the method was able to analyze plasma and cerebrospinal fluid samples. This work improves the utility of benzoyl chloride, which labels multiple important functional groups, for widely targeted metabolomics methods. Neuropeptides constitute the largest group of neurotransmitters in the central nervous system. Neuropeptide signaling is involved in many physiological functions but detection in vivo is challenging due to low picomolar concentrations. Targeted capillary LC-MS methods were developed for neurotensin, oxytocin, dynorphin, and enkephalins. The assays utilize desalting and preconcentration on a single analytical column to achieve low picomolar limits of detection. Detection was improved by optimizing all facets of neuropeptide handling from sampling to detection with capillary LC-MS. These techniques were applied to examine several aspects of neuronal function. Specific neuronal circuits were defined, confirmed, and targeted by combining these analytical tools with pharmacogenetic and optogenetic methods. Novel pathophysiological changes to opioid neuropeptide (dynorphin and enkephalins) dynamics were elucidated in a rat model of Parkinson’s disease, and a potential neuropeptide-based treatment was established to reduce the abnormal dyskinetic movements associated with chronic dopamine replacement therapies in Parkinson’s Disease. These new multiplexed approaches will advance our understanding of the complex processes underlying neuronal function at the molecular and circuit levels, as well as provides an improved set of experimental tools to better understand lingering questions in the field of neuroscience.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135792/1/jmtwong_1.pd

Theranostics of Glioblastoma Multiforme: In Vitro Characterization of Targeted Nanoemulsions and Creation of a 3D Statistical Heatmap to Visualize Nanoemulsion Uptake

Glioblastoma Multiforme (GBM) continues to be a leading form of malignant brain cancer. GBM presents a high variability patient to patient. This provides a need for a theranostic system capable of both delivering therapeutic and in real-time visually observe results. Current methods involve months between treatment application and available results. Based on Nemucore Medical Innovation’s drug delivery vehicles, nanoemulsions (NE) containing chemotherapeutics are being utilized to target cancer cells and reduce systemic toxicity. Through the automation of required statistical analysis and creation of a wire-frame rat brain model with 174 defined brain regions a software capable of providing ‘real-time’ pharmacodynamics analysis of Gd3+ annotated, receptor-targeted NE is one step closer

Molecular mechanisms of nociception and pain

My thesis uses in vivo calcium imaging to investigate the cell and molecular mechanisms of two unusual pain states: congenital analgesia and cold allodynia. Genetic deletion of voltage-gated sodium channel NaV1.7 in mice and humans leads to profound pain insensitivity. Paradoxically, peripherally-targeted pharmacological antagonists of NaV1.7 fail to relieve pain in the clinic. To determine the mechanism of analgesia in NaV1.7 null mutants, I used optical, electrophysiological and behavioural methods to investigate the effect of peripheral NaV1.7 deletion on nociceptor function. Surprisingly, both calcium imaging and extracellular recording of NaV1.7-deficient sensory neurons in vivo found limited deficits in the response to noxious stimuli. Synaptic transmission from nociceptor central terminals in the spinal cord was however compromised following NaV1.7 deletion. Importantly, both synaptic deficits and behavioural analgesia were reversed by blocking central opioid receptors. Collectively, these data account for the failure of peripherally-targeted NaV1.7 blockers and point to a central mechanism of analgesia in NaV1.7 null mutants that requires opioid receptors. Chronic pain patients suffering from cold allodynia experience normally innocuous cooling as excruciating pain, but the cells and molecules driving cold allodynia remain elusive. I used in vivo calcium imaging to investigate how the activity of cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, peripheral nerve injury and ciguatera poisoning. In neuropathic mice exhibiting cold allodynia, a subset of cold-insensitive, large-diameter, peptidergic nociceptors became responsive to cooling. Diptheria toxin-mediated ablation of these silent cold-sensing neurons decreased neuropathic cold hypersensitivity. Voltage-gated potassium channels KV1.1 and KV1.2 were highly expressed in silent cold-sensing neurons and pharmacological inhibition of these channels rapidly induced cold responsiveness in cold-insensitive neurons. Taken together, I reveal that silent-cold sensing neurons contribute to cold allodynia in neuropathic pain and identify KV1 channel downregulation as a driver of de novo cold sensitivity, in vivo

Applications and Experiences of Quality Control

The rich palette of topics set out in this book provides a sufficiently broad overview of the developments in the field of quality control. By providing detailed information on various aspects of quality control, this book can serve as a basis for starting interdisciplinary cooperation, which has increasingly become an integral part of scientific and applied research

Preclinical Animal Modeling in Medicine

The results of preclinical animal research have been successfully implemented in various medical and biological practices. The use of animals in medicine is based on significant anatomical, physiological, and molecular similarities between humans and animals. Particularly, mammals that have vast biological commonalities with humans represent not only a valuable model to explore the mechanisms of varied human diseases, but also to define new diagnostic and treatment strategies. This book covers broad but important aspects of animal modeling for scientific medicine as well as for translational systems and biological sciences. Alternative methods such as cell culture and in vitro experiments that do not require the sacrifice of an animal are encouraged for scientific and medical studies

Recent Advances in Minimally Invasive Surgery

Minimally invasive surgery has become a common term in visceral as well as gynecologic surgery. It has almost evolved into its own surgical speciality over the past 20 years. Today, being firmly established in every subspeciality of visceral surgery, it is now no longer a distinct skillset, but a fixed part of the armamentarium of surgical options available. In every indication, the advantages of a minimally invasive approach include reduced intraoperative blood loss, less postoperative pain, and shorter rehabilitation times, as well as a marked reduction of overall and surgical postoperative morbidity. In the advent of modern oncologic treatment algorithms, these effects not only lower the immediate impact that an operation has on the patient, but also become important key steps in reducing the side-effects of surgery. Thus, they enable surgery to become a module in modern multi-disciplinary cancer treatment, which blends into multimodular treatment options at different times and prolongs and widens the possibilities available to cancer patients. In this quickly changing environment, the requirement to learn and refine not only open surgical but also different minimally invasive techniques on high levels deeply impact modern surgical training pathways. The use of modern elearning tools and new and praxis-based surgical training possibilities have been readily integrated into modern surgical education,which persists throughout the whole surgical career of modern gynecologic and visceral surgery specialists

DEVELOPMENT AND APPLICATION OF ANALYTICAL METHODS FOR FENTANYL ANALOGS IN TRADITIONAL AND ALTERNATIVE MATRICES

Over the last decade, the United States has experienced increasing numbers of overdose deaths attributed to opioid abuse. While the opioid crisis originated with prescription opioids, recent impacts are credited to synthetic novel psychoactive substances (NPS). Synthetic opioids, a category of NPS, can be divided into two categories: fentanyl- and non-fentanyl derivatives. Fentanyl analogs, the focus of this research, not only pose a significant threat to public safety, but also challenges to forensic laboratories due to their high potency, low concentrations, similar molecular structures, and progressive prevalence. To address detection issues faced by forensic toxicologists, it is necessary to develop highly sensitive analytical methods for detecting fentanyl analogs in traditional and alternative biological matrices. The goals of this study were to 1) develop and validate a data-independent screening method for fentanyl analogs in whole blood and oral fluid for application to postmortem specimens and antemortem oral fluid collected from detainees, respectively; 2) develop and validate a quantitative method for furanyl fentanyl and its metabolites in human and rat plasma for future pharmacological assessment; 3) develop and validate a quantitative method for prevalent fentanyl analogs in whole blood and perform a long-term stability study; and 4) develop and validate a quantitative method for fentanyl analogs in oral fluid for application to antemortem oral fluid samples collected from probationers/parolees. A data-independent screening method was developed and validated for fentanyl analogs (n=14) in whole blood and oral fluid using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Data were acquired in time of flight (TOF) and All Ions fragmentation (AIF) modes and low limits of detection were achieved. A personal compound database and library (PCDL) was developed for targeted and exogenous compound identification. Postmortem blood samples (n=30) received from National Medical Services (NMS) Labs and oral fluid samples (n=20) collected from detainees in Texas detention centers were screened for fentanyl analogs. In the blood samples, analogs of furanylfentanyl (n=16), 4-ANPP (n=16), cis-3-methylfentanyl (n=4), fentanyl (n=4), norfentanyl (n=2), and valerylfentanyl (n=1) were detected. No fentanyl analogs were detected in the oral fluid samples. A quantitative method was developed and validated for furanylfentanyl and its metabolites (4-ANPP and furanyl norfentanyl) in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using American National Standards Institute/American Standards Board (ANSI/ASB) Standard 036: Standard Practices for Method Validation in Forensic Toxicology. Low limits of detection and small sample volumes (100 μL) were achieved. The method was cross validated in rat plasma for potential application to a pre-clinical pharmacodynamic/pharmacokinetic (PD/PK) study. A method was developed and validated for the quantification of prevalent fentanyl analogs (n=13) in blood using targeted data acquisition on an LC-QTOF-MS. The method was validated according to ANSI/ASB Standard 036. The method was applied to a long-term stability study assessing fentanyl analog degradation over 9 months at four temperature conditions (-20⁰C, 4⁰C, 25⁰C, and 35⁰C). Results described minimal instability under room temperature and refrigerated storage, degradation after 4 freeze/thaw cycles, and instability after 1 week of elevated exposure. Acrylfentanyl had a high degree of instability under most temperature conditions and breakdown mechanism remains undetermined. Authentic forensic blood specimens stored under refrigeration were analyzed 6 months apart to assess stability in postmortem samples. Furanylfentanyl (n=4) and 4-ANPP (n=7) were quantifiable and exhibited percent loss of 0.2-26.8% and 16.3-37.4%, respectively. Loss was attributed to sample source, age, and composition. The aforementioned data acquisition was utilized to develop and validate a quantification method for fentanyl analogs (n=13) in oral fluid using LC-QTOF-MS by ANSI/ASB Standard 036. The method was applied to authentic oral fluid samples (n=16) received from Redwood Toxicology obtained from probationers/parolees. Oral fluid samples were positive for fentanyl (n=16) and 4-ANPP (n=3) at concentrations of 1.0-104.5 ng/mL and 1.2-5.7 ng/mL, respectively. No fentanyl analogs were detected. The present work describes sensitive analytical methods for the detection and quantification of fentanyl analogs with proven applicability to forensically relevant samples. In addition, challenges associated with analyte detection, compound differentiation, and drug instability have been addressed. With the constant emergence of novel fentanyl analogs, forensic toxicologists must be proactive with advancement of analytical analyses and sample treatment

Proceedings of the 97th Annual Virginia Academy of Science Meeting, 2019

Proceedings of the 97th Annual Virginia Academy of Science Meeting, May 22-24, 2019, at Old Dominion University, Norfolk, Virginia

Research and Creative Activity, July 1, 2020-June 30, 2021: Major Sponsored Programs and Faculty Accomplishments in Research and Creative Activity, University of Nebraska-Lincoln

Foreword by Bob Wilhelm, Vice Chancellor for Research and Economic Development, University of Nebraska-Lincoln: This booklet highlights successes in research, scholarship and creative activity by University of Nebraska–Lincoln faculty during the fiscal year running July 1, 2020, to June 30, 2021. It lists investigators, project titles and funding sources on major grants and sponsored awards received during the year; fellowships and other recognitions and honors bestowed on our faculty; books and chapters published by faculty; performances, exhibitions and other examples of creative activity; patents and licensing agreements issued; National Science Foundation I-CORPS teams; and peer-reviewed journal articles and conference presentations. In recognition of the important role faculty have in the undergraduate experience at Nebraska, this booklet notes the students and mentors participating in the Undergraduate Creative Activities and Research Experience (UCARE) and the First-Year Research Experience (FYRE) programs. While metrics cannot convey the full impact of our work, they are tangible measures of growth. A few achievements of note: • UNL achieved a record $320 million in total research expenditures in FY 2020, a 43• Our faculty earned 1,508 sponsored research awards in FY 2020. University-sponsored industry activity also spurred economic growth for Nebraska. • Nebraska Innovation Campus created 1,948 jobs statewide and had a total economic impact of$372 million. • Industry sponsorship supported $19.2 million in research expenditures. • NUtech Ventures brought in$6.48 million in licensing income. I applaud the Nebraska Research community for its determination and commitment during a challenging year. Your hard work has made it possible for our momentum to continue growing. Our university is poised for even greater success. The Grand Challenges initiative provides a framework for developing bold ideas to solve society’s greatest issues, which is how we will have the greatest impact as an institution. Please visit research.unl.edu/grandchallenges to learn more. We’re also renewing our campus commitment to a journey of anti-racism and racial equity, which is among the most important work we’ll do. I am pleased to present this record of accomplishments. Contents Awards of $5 Million or More Awards of$1 Million to $4,999,999 Awards of$250,000 to $999,999 50 Early Career Awards Arts and Humanities Awards of$250,000 or More Arts and Humanities Awards of $50,000 to$249,999 Arts and Humanities Awards of $5,000 to$49,999 Patents License Agreements National Science Foundation Innovation Corps Teams Creative Activity Books Recognitions and Honors Journal Articles 105 Conference Presentations UCARE and FYRE Projects Glossar