Genetic approaches to understanding pain mechanisms: Zfhx2 and peripheral sensory neuron ablation mouse transgenic models

Latest cutting-edge sequencing has allowed researchers to obtain a full array of differentially expressed neuronal genes within the peripheral nervous system. Understanding this heterogeneity and functional implication could unveil new therapeutic targets towards a more precise medicine. Combining a novel reporter mouse with Cre recombinase strategies, I examined the spatial and functional organization of transcriptomically different subpopulations of neurons in the mouse DRG in pathological and nonpathological states. Results herein include: confirmation of Cre activity and specificity in all lines studied by RNA scope when compared to previous reports and transcriptomic analysis; significant upregulation of DRG gal expression after Complete Freund's Adjuvant (CFA) induced inflamation; normal weight and exploratory behaviour for all lines tested; motor activity assessed by Rotarod not significant, but further motor coordination tests on animals missing Th DRG neurons showed significant impairment; noxious mechanosensation reduced in animals lacking SCN10aCre and Tmem45b DRG; confirmation of CGRP-positive neurons role in heat and cold perception as well as in the formalin inflammatory model; Von Frey hypersensitivity on animals lacking CGRP; and lastly TrkBpositive neurons responsible for significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model whilst no effect in cancer induced bone pain model. Parallelly, by reverse genetics approach, I explore the contribution of the Zfhx2 gene, whose mutation has been identified as responsible for the Marsili pain insensitivity syndrome, in two different animal models of nociception. Behavioural characterisation of bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine mutation, as well as Zfhx2 null mutant mice, shows significant deficits in pain sensitivity in thermal and mechanical tests respectively. In summary, as well as validating several new useful transgenic mouse lines, this thesis provides insights into genes and neuronal subpopulations important in pain pathways and provides potential platforms for translational studies of pain syndromes

Molecular basis of FAAH-OUT-associated human pain insensitivity

Chronic pain affects millions of people worldwide and new treatments are needed urgently. One way to identify novel analgesic strategies is to understand the biological dysfunctions that lead to human inherited pain insensitivity disorders. Here we report how the recently discovered brain and dorsal root ganglia-expressed FAAH-OUT long non-coding RNA (lncRNA) gene, which was found from studying a pain-insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme. We demonstrate that the disruption in FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression. Furthermore, using transcriptomic analyses in patient-derived cells we have uncovered a network of genes that are dysregulated from disruption of the FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed. Given that FAAH is a potential target for the treatment of pain, anxiety, depression and other neurological disorders, this new understanding of the regulatory role of the FAAH-OUT gene provides a platform for the development of future gene and small molecule therapies

Sensory neuron–derived NaV1.7 contributes to dorsal horn neuron excitability

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno–electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system–specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis

Molecular basis of FAAH-OUT-associated human pain insensitivity.

Chronic pain affects millions of people worldwide and new treatments are needed urgently. One way to identify novel analgesic strategies is to understand the biological dysfunctions that lead to human inherited pain insensitivity disorders. Here we report how the recently discovered brain and dorsal root ganglia-expressed FAAH-OUT long non-coding RNA (lncRNA) gene, which was found from studying a pain-insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme. We demonstrate that the disruption in FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression. Furthermore, using transcriptomic analyses in patient-derived cells we have uncovered a network of genes that are dysregulated from disruption of the FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed. Given that FAAH is a potential target for the treatment of pain, anxiety, depression and other neurological disorders, this new understanding of the regulatory role of the FAAH-OUT gene provides a platform for the development of future gene and small molecule therapies.- Medical Research Council/United Kingdom - grant No. [G1100340/MRC_]. - Medical Research Council/United Kingdom - grant No. [MR/R011737/1/MRC_]. - Versus Arthritis/United Kingdom [20200/VAC_]. - Medical Research Council grant [G1100340]. - Medical Research Council grant [MR/R011737/1]. - Qatar University grants [QUSD-CMED-2018/9-3] and [QUCG-CMED-19/20-4]. - Qatar National Research Fund [NPRP13S-0209-200315]. - Versus Arthritis grant [20200]. - Wellcome grant [200183/Z/15/Z]

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Sensitization of Cutaneous Primary Afferents in Bone Cancer Revealed by In Vivo Calcium Imaging

Cancer-induced bone pain (CIBP) is a complex condition, comprising components of inflammatory and neuropathic processes, but changes in the physiological response profiles of bone-innervating and cutaneous afferents remain poorly understood. We used a combination of retrograde labelling and in vivo calcium imaging of bone marrow-innervating dorsal root ganglia (DRG) neurons to determine the contribution of these cells in the maintenance of CIBP. We found a majority of femoral bone afferent cell bodies in L3 dorsal root ganglia (DRG) that also express the sodium channel subtype Nav1.8&mdash;a marker of nociceptive neurons&mdash;and lack expression of parvalbumin&mdash;a marker for proprioceptive primary afferents. Surprisingly, the response properties of bone marrow afferents to both increased intraosseous pressure and acid were unchanged by the presence of cancer. On the other hand, we found increased excitability and polymodality of cutaneous afferents innervating the ipsilateral paw in cancer bearing animals, as well as a behavioural phenotype that suggests changes at the level of the DRG contribute to secondary hypersensitivity. This study demonstrates that cutaneous afferents at distant sites from the tumour bearing tissue contribute to mechanical hypersensitivity, highlighting these cells as targets for analgesia

Työyhteisön suorituskyvyn kehittäminen : case: Tilitoimisto Oy

Tämän opinnäytetyön aiheena on työyhteisön suorituskyvyn kehittäminen tilitoimistossa. Työ keskittyy antamaan kohdeyritykselle keinoja, joiden avulla toimintaa pystyttäisiin kehittämään ja tehostamaan. Opinnäytetyön tavoitteena on selvittää, miten työntekijöiden ja työyhteisön suorituskykyä voidaan kehittää tilitoimiston näkökulmasta ajateltuna. Oppinäytetyössä perehdytään suorituskyvyn muodostumiseen ja sen ylläpitämiseen, sekä tarkastellaan suorituskyvyn vaikutuksia yrityksen kilpailukykyyn ja tuottavuuteen. Työskentely tilitoimistossa on haastavaa jatkuvan kiireen ja määräpäivien vuoksi. Toiminnan laadun turvaamiseksi tilitoimistossa on syytä panostaa myös työntekijöihin työyhteisön toimintaa ja työhyvinvointia kehittämällä. Suorituskykyyn vaikuttavat monet yksilö- ja yhteisötason tekijät. Näiden ollessa tasapainossa voidaan työyhteisössä saavuttaa työhyvinvointia ja näin ollen parantaa työyhteisön tuottavuutta ja tehokkuutta. Työn teoriaosuus on jaettu kolmeen osaan; tilitoimistoalan kuvaaminen, suorituskyvyn muodostuminen ja työhyvinvoinnin vaikutukset suorituskykyyn ja tuottavuuteen. Empiriaosuuden aineisto hankittiin työntekijöille tehdyn kyselyn ja ajankäytön seurannan avulla, lisäksi empiriaosuudessa on tuotu esiin omia havaintojani aiheesta. Empiriaosuuden tulokset osoittavat, että työyhteisön toiminnoissa on kehittämistä. Tuloksista selviää, että työyhteisön tiedonkulussa ja sisäisessä koulutuksessa on aihetta kehittämiseen. Työntekijöiden hyödyntäminen toiminnan suunnittelussa ja kehittämisessä olisi myös tärkeää. Työntekijöiden työaikaa kuluu suurissa osin laskutettaviin tehtäviin, mutta aikavarkaitakin löytyy. Näihin lukeutuvat työn keskeytymiset, keskustelut asiakkaiden kanssa, neuvojen kysyminen toiselta työntekijältä sekä toisaalta toisten työntekijöiden neuvonta. Jatkotutkimuksena voisi selvittää, kuinka hyvin suorituskyvyn ja työyhteisön kehittäminen on vaikuttanut toiminnan tehokkuuteen. Lisäksi voitaisiin verrata työntekijöiden ajankäyttöä sekä todellisuudessa asiakkailta laskutettavia tunteja.The subject of this thesis is the improvement of efficiency and employee performance in an accounting company. The thesis focuses on giving advices to the target company on how develop and improve employee performance. The aim of this thesis is to clarify how the employees’ and the work community’s performance could be developed from accounting company’s point of view. This thesis therefore focuses on the formation of performance and how to maintain it but also on the impacts performance may have on competitiveness and productivity. Working in an accounting company is challenging because of constant rush and deadlines. To secure the quality of performance an accounting company should invest in employees by developing well-being and performance at work. Performance is affected by many factors both at individual level and at the level of the whole work community. When these factors are in balance, the work community may achieve well-being and thereby improve its productivity and efficiency. The theoretical part of the thesis is divided into three parts. First, this section describes the field of accounting. Then, it discusses how performance is formed. Finally, this section examines how well-being affects performance and productivity. The empirical material is collected by employee survey and time tracking but also by my own notices. Based on the results of this study, there is need for improvement in the work community. The results shows that internal communication, training and the overall work climate need improvement. Working time is mainly spent productively, but there are also time stealers such as interruptions, conversations with clients and asking and giving advice to co-workers. Further study could determine, how improving performance and developing the work community have affected productivity. Moreover, employees’ working hours could be compared with the actual chargeable hours

MicroRNA-1-associated effects of neuron-specific brain-derived neurotrophic factor gene deletion in dorsal root ganglia

MicroRNAs (miRNAs) regulate gene expression in physiological as well as in pathological processes, including chronic pain. Whether deletion of a gene can affect expression of the miRNAs that associate with the deleted gene mRNA remains elusive. We investigated the effects of brain-derived neurotrophic factor (Bdnf) gene deletion on the expression of miR-1 in dorsal root ganglion (DRG) neurons and its pain-associated downstream targets heat shock protein 60 (Hsp60) and connexin 43 (Cx43) in tamoxifen-inducible conditional knockout mice, Bdnf(fl/fl); Advillin-CreER(T2) (Bdnf cKO). Efficient Bdnf gene deletion was confirmed in DRG of Bdnf cKO mice by Real-Time qRT-PCR and ELISA 10days after completed tamoxifen treatment. In DRG, miR-1 expression was reduced 0.44-fold (p <0.05; Real-time qRT-PCR) in Bdnf cKO compared to floxed wildtype littermate control Bdnf(fl/fl) mice (WT). While Hsp60 protein expression was increased 1.85-fold (p <0.05; Western blot analysis), expression levels of Cx43 and the miR-1-associated transcription factors MEF2a and SRF remained unchanged. When analyzing Bdnf cKO mice 32days after complete tamoxifen treatment to investigate whether observed expression alterations remain permanently, we found no significant differences between Bdnf cKO and WT mice. However, miRNA microarray analysis revealed that 167 miRNAs altered (p <0.05) in DRG of these mice following Bdnf gene deletion. Our results indicate that deletion of Bdnf in DRG neurons leads to a temporary dysregulation of miR-1, suggesting an impairment of a presumable feedback loop between BDNF protein and its targeting miR-1. This appears to affect its downstream protein Hsp60 and as a consequence might influence the phenotype after inducible Bdnf gene deletion. While this appears to be a MEF2a-/SRF-independent and transient effect, expression levels of various other miRNAs may remain permanently altere