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Proteasome Inhibitors : a novel therapy that blunt hyperglycemia-induced cardiac contractile dysfunctionAdams, Buin 04 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Diabetes is considered a major threat to human health in both developed and developing nations. Cardiovascular disease which is common in diabetic patients has increased the overall disease affliction. Moreover, stress-induced hyperglycemia has led to increased mortality and morbidity in patients with an acute myocardial infarction (MI), whether the patient has diabetes or not. In addition, acute MI might stem from stress-induced hyperglycemia capability to increase inflammation and oxidative stress resulting in a worse functional cardiac outcome. Hyperglycemia-induced oxidative stress can similarly result in the formation of miss folded or damaged proteins that may be eliminated by the ubiquitin-proteasome system (UPS).
Futhermore, hyperglycemia-induced oxidative stress can also result in dysregulation of the UPS that removes these misfolded proteins. Additionally, an increasing body of evidence implicates UPS dysfunction in cardiac diseases and hyperglycemia which has been associated with increased inflammation and blunted cardiac function in response to ischemia-reperfusion. Literature however is blurred whether a reduction or a rise in the UPS is damaging with hyperglycemia and in response to ischemia-reperfusion. In light of this, we hypothesized that UPS inhibitors such as Z-Leu-Leu-Leu-al (MG-132) and lactacystin, protects the rat heart against ischemia-reperfusion under hyperglycemic perfusion conditions.
Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 minutes reperfusion ± PI treatment (MG-132 and lactacystin), anti-inflammatory (Ibuprofen) and anti-oxidant (NAC). Infarct size was determined using Evans Blue dye and 1% 2,3,5-triphenyl tetrazolium chloride (TTC) staining with 20 minutes regional ischemia and 2 hours reperfusion ± PI’s treatments. Tissues were collected at the end of the global ischemia experiments and analyzed for UPS activity, oxidative stress, apoptosis and inflammation.
Our data expressed a reduced cardiac contractile function in response to ischemia and reperfusion under hyperglycemic conditions as well as an increase in UPS activity. PI treatment resulted in cardio-protection for ex vivo rat heart model exposed to ischemia and reperfusion under hyperglycemic conditions as well as ibuprofen and NAC. In parallel lactacystin treatment significantly decreased myocardial oxidative stress, apoptosis, and inflammation which provided cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions
This study shows that acute hyperglycemia elicits myocardial oxidative stress, apoptosis and inflammation that in time results in an increase in contractile dysfunction following ischemia and reperfusion. However, we found that PI treatment with both MG-132 and lactacystin blunted high glucose-induced damaging effects which resulted in a robust cardio-protection in response to ischemia and reperfusion under hyperglycemic conditions, by reducing oxidative stress, decreasing apoptosis and limiting inflammation. A parallel outcome was observed at baseline although the underlying mechanisms driving this process still need to be clarified. Our findings indicate that the UPS may be a unique therapeutic target to treat ischemic heart disease in diabetic patients, and non-diabetic individuals that present with stress-induced hyperglycemia. In summary, this thesis established that PIs act as a novel cardio-protective intervention to treat acute hyperglycemia with associated cardiovascular complications. / AFRIKAANSE OPSOMMING: Diabeties word beskou as ‘n baie groot problem vir menslieke gesondhied vir biede die ontwikkel en onontwikkelende lande. Kardiovaskulêre siekte wat normaal met diabetiese pasiente geassoseerd word veroorsaak ‘n toeneemende druk, wat hierdie siekte laat toeneem. Verder meer vergroot stresgeïnduseerde hiperglukemie die mortaliteit van pasiente met of sonder diabeties wat akute miokardiale infarksie onder lede het. Akute miokardiale infarksie kan ook ontstaan van stresgeïnduseerde hiperglukemie se bekwaamheid om meer inflamasie en oksidante stress te veroorsaak wat in ‘n meer swakker funksionele kardiale toestand. Hiperglukemiegeïnduseerde oksidatiewe stres ook tot wanregulering van die ubikwitien-proteosoomsisteem (UPS) wat wangevoude protïene verwyder, aanleiding gee. Kontrasterende data bestaan van verhoogde/verlaagde UPS aktiwietiet, sowel as met hiperglukemie en/of in reaksie tot isgemie-reperfussie. As gewolg hiervan,, hipotetiseer ons dat Z-Leu-Leu-Leu-al (MG-132) and lactacystin as ‘n nuwe kardiobeskermingsmiddel kan optree deur miokardiale oksidatiewe stress, inflamasie en UPS aktiwiteit te verlaag in reaksie op isgemie-reperfussie tydens akute hiperglukemiese toestande kan verlaag.
Geïsoleerde rotharte is ex vivo met Krebs-Henseleit buffer, wat, 33 mM glukose vs. kontrole (11 mM glukose) bevat, vir 60 min geperfuseer, daarna is dit deur 20 min globale isgemie gevolg en 60 min reperfussie ± PI behandeling (MG-132 and lactacystin), antiflammatoriese behandeling (Ibuprofen) en antioxidant behandeling (NAC). Infarkgrootte is bepaal deur Evans bou kleursel en 1% 2. 3-5 tripfeniel tetrazoloimcholierd (TTC) kleuring met 20 minute regionale ischemie, en 2 uur reprefussie ± PI’s behandeling. Weefsels is aan die einde van die globale isgemie eksperimente versamel, en vir oksidatewe stres, apoptose en inflammasie ontleed.
Ons data toon aan dat kardiale kontraktiele funksie in reaksie op isgemie-reperfussie onder hiperglukemiese toestande verlaag het asook ‘n toename in UPS aktiwitiet veroorsaak. PI behandeling het gelei tot kardiale beskerming vir ex vivo rotharte wat aan isgemie-reperfussie onder hiperglukemiese toestande blootgestel was sowel as ibuprofen en NAC. Parallel hiermee het lactacystin oksidatiewe stres, apoptose, inflmasie, en UPS aktiwiteit na isgemie-reperfussie, verlaag in reaksie isgemie-reperfussie onder hiperglukemiese toestande.
Hierdie studie het bevind dat akute hiperglukemie, miokardiale oksidatiewe stres lei tot oksidante stress, apoptose, en inflamasie na kontraktiele wanfunksionering na isgemie-reperfussie lei. Ons het bevind dat beide MG-132 en lactacystin behandeling, hoë glukose-geïnduseerde skadelike effekte onderdruk, en kardiale-beskerming in reaksie op isgemie-reperfussie onder hiperglukemiese toestande ondervind was deur oksidante stress, apoptose, en inflamasie te verlaag. ‘n Soorgelyke effek is tydens die basislyn waargeneem, alhoewel die onderliggende meganisme wat hierdie proses meer ondersoek instel. Ons bevinding dei dat die UPS ‘n nuwe behandeling teiken kan word in sgemie-geïnduseerde reperfussie onder aktute en chroniese hoë glukose toestande. In opsomming, het die tesis belowend bevindinge gevind wat ‘n nuwe terapeutiese intervensie vir die behandeling van akute hiperglukemie met geassosieërde kardiovaskulêre komplikasies gebruik kan word.
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Insight Into Autonomic Dysfunctions With Novel Interventions: Focusing On Vascular Tone And Breathing RegulationsZhang, Shuang 09 May 2016 (has links)
The autonomic nervous system (ANS) controls most involuntary functions of the body. Dysfunctions of the ANS can be life-threatening. However, several critical questions related to cardiovascular and breathing regulations remain unclear.
One of the open questions is how the system lose control of the vascular tones under certain circumstances. Using the septic shock model induced by lipopolysaccharide (LPS) in isolated and perfused mesenteric arterial rings, we found the vascular hyporeactivity is attributed to the decreased vasoconstriction to α-adrenoceptor agonists. The endotoxin-induced vasodilation can be intervened with endothelin-1 (ET-1), serotonin (5-HT) or vasopressin, which have never been used in clinical treatment.
It is unclear how the excitability of endothelium affects vascular tones. Using optogenetics and transgenic mice with channelrhodopsin expression in endothelial cells (ECs), we found selective activation of the ECs induces a fast, robust, reproducible and long-lasting vasoconstriction in isolated and perfused hearts and kidneys.
Breathing control by the ANS within the brain becomes abnormal in certain genetic diseases, such as Rett syndrome with defected norepinephrine (NE) system in locus coeruleus (LC). The LC neurons are hyperexcitable while NE release is deficient. Using optogenetics and double transgenic mice with Mecp2 null and channelrhodopsin expression in LC neurons, we found the NE-ergic modulation of hypoglossal neurons was impaired in transgenic mice, which cannot be improved with optostimulation, suggesting that LC neuronal hyperexcitability may not benefit the NE modulation in Rett syndrome.
Collectively, our results provide insight into the autonomic dysfunctions using experimental interventions that have barely been used before.
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DIFFERENCES IN DIMENSIONS OF CHILDHOOD FUNCTIONING IN CHILDREN OF PRETERM VERSUS FULL TERM BIRTH STATUSTurner, Tameika Shenay 01 January 2006 (has links)
As medical advances are made in the area of neonatology, more and more premature babies are surviving at younger gestational ages and lower birth weights. Growth in the survival rates of preterm infants leads to questions regarding the long term developmental trajectory of these children. The current study sought to expand on research regarding dimensions of childhood functioning and to apply it to the problem of prematurity by (a) utilizing a new instrument: the Merrill Palmer Revised edition, (b) including children of preterm and full term birth statuses from as young as 2 months of age, and (c) collecting data from parental and clinician reports. In addition to attempts to clarify the relationship between birth status and childhood dysfunction, this study also sought to augment existing literature by exploring the correlation between parental report and clinician observation of childhood dysfunction. The results of this study did not support the hypothesis that children of preterm birth will demonstrate more problems in functioning when compared to full term peers. Although there were more significant differences between preterm and full term children in the older cohort group, those differences did not consistently reflect dysfunction by the preterm children. Additionally, this study considered dimensions of dysfunction as measured by parental report and clinician observations. Notably, a lack of agreement between parent and clinician observations emerged for the young age cohort group. However, the high level of agreement for the older children suggests that parental and clinician perspectives converge with older children. Contrary to the hypothesis, birth status, gender, ethnicity, and SES did not collectively form a specific risk index for dysfunction. However, these factors did interact with each other to predict functioning on several scales. In fact, there were no significant main effects. Instead, predictors of dysfunction were interactions of variables such as birth status, age, gender, and ethnicity. This general finding illustrates the importance of taking into consideration all aspects of the childs situation when making an assessment of functioning.
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Otolith Dysfunction and Postural Stability: A Chronic Effects of Neurotrauma Consortium StudyAkin, Faith, Hall, Courtney D. 01 August 2018 (has links)
No description available.
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Análise da relação entre estresse ocupacional, sinais e sintomas de DTM e atividade eletromiográfica dos músculos mastigatórios em militares da 5ª CSM de Ribeirão Preto/SP / Relation between occupational stress, TMD signs and symptoms and electromyographic activity of masticatory muscles in military personnel of the 5th CSM of Ribeirão Preto, BrazilSilva, Carina Thaís de Almeida e 15 June 2012 (has links)
Atualmente, o nível de exigências do mercado de trabalho tem aumentado, a qualificação profissional se tornou indispensável, a concorrência está cada vez maior e por isso, o estresse ocupacional tem destaque no processo de saúde-doença do individuo em seu ambiente de trabalho. O mecanismo interno de liberação de estresse pode causar, entre outros, um aumento na tonicidade da musculatura da cabeça e do pescoço, como também dos níveis de atividade muscular parafuncional, como o bruxismo e o apertamento dentário. Assim, o cirurgião-dentista tem o papel de diagnosticar, tratar e proservar os pacientes com disfunção temporomandibular. Uma das profissões mais históricas no Brasil, ser militar, especificamente da força armada terrestre, o Exército Brasileiro tem, além de suas características de honestidade, vigor físico, responsabilidade e pontualidade, fatores associados que podem gerar o estresse ocupacional, como o risco à vida, sujeição a preceitos rígidos de hierarquia e disciplina, disponibilidade permanente, mobilidade geográfica, formação específica e aperfeiçoamento constante, além das consequências para a família. Nesse contexto, o objetivo desta pesquisa foi investigar a presença de estresse ocupacional em uma população de militares no município de Ribeirão Preto, Estado de São Paulo, Brasil e analisar sua relação com os achados clínicos de DTM e o exame eletromiográfico dos músculos mastigatórios. A amostra foi composta por 30 sujeitos da pesquisa, militares da 5ª CSM. A presença de DTM foi investigada por meio de questionário anamnésico (ProDTMMulti) e de exame clínico odontológico (RDC/TMD; Helkimo). O estresse ocupacional foi investigado por meio de questionário específico, Maslach Burnout Inventory (MBI). Os exames eletromiográficos e de força de mordida foram realizados no gabinete odontológico da 5ª CSM por cirurgiã-dentista experiente, a qual participa ativamente do serviço como profissional militar contratada pela instituição. Os resultados mostraram que no exame clínico, 90% dos militares não foi classificável pelo RDC e apenas 20% foram classificados com disfunção severa (grupo 3) do Índice de HELKIMO. Pelo Protocolo ProDTMMulti: 46,66% dos militares se enquadraram no grau zero (ausência de DTM), 40% no grau 1 (DTM leve) e apenas 13,33% dos militares relataram sintomas um pouco mais acentuados, ficando no grau 2 (DTM moderada). Com relação ao estresse, a única correlação existente foi com a patente: quanto maior a patente, menor o estresse. Pela análise eletromiográfica nos testes estáticos e dinâmicos da mandíbula, observou-se simetria entre os lados direito e esquerdo, e um padrão mastigatório equilibrado entre os lados. No teste de força máxima de mordida, a força também se apresentou equilibrada entre os lados. Concluiu-se que o estresse ocupacional está presente nesta amostra, porém não se correlacionou com os achados clínicos de DTM e com o exame eletromiográfico dos músculos mastigatórios dos militares da 5ª CSM do município de Ribeirão Preto/SP. / As the labor market requirements have increased, professional qualification has been taken as essential and the work competition keeps heightening, occupational stress has become prominent in the health-disease process of the working person. Among other things, the internal mechanism of stress release can cause an increased tonicity of head and neck muscles and increased levels of muscular parafunctional activity such as teeth grinding (bruxism) and teeth clenching. The dentist has the role in diagnosing, treating and following-up patients with temporomandibular dysfunction. One of the most historic occupations in Brazil, the military career at the Brazilian Army, besides characteristics of honesty, physical force, responsibility and punctuality, has associated factors that can lead to occupational stress, as the risk of death, to be under precepts of strict discipline and hierarchy, permanent availability, geographic mobility, specific training and continuous improvement, and the resulting consequences for the family. The objective of this research was to study the relation between occupational stress and degree of TMD in soldiers of Ribeirão Preto, State of São Paulo, Brazil. The study sample had 30 military volunteers from the 5th CSM. The presence of TMD was investigated by use of an anamnestic questionnaire (ProDTMMulti) and dental clinical examination (RDC/TMD; Helkimo). Occupational stress was investigated by means of a specific questionnaire, Maslach Burnout Inventory (MBI). Electromyographic and bite force tests were held in the dental office of the 5ª CSM by a qualified dentist who participates actively in the service as a military professional hired by the institution. Results of clinical examination showed that 90% of the studied subjects were not classifiable by the RDC and only 20% were classifiable as having severe dysfunction (Group 3) by HELKIMO Index. By the ProDTMMulti Protocol, 46,66% of the individuals were classified in Grade 0 (absence of TMD), 40% in Grade I (light TMD) and only 13,33% reported symptoms a little more pronounced being in Grade II (moderate TMD). Regarding stress, the only identified correlation was with the military rank: higher the rank, lower the stress. By electromyography analysis in mandible static and dynamic tests, it was observed symmetry between right and left sides, and a balanced masticatory pattern between sides. In the maximal bite force test, the force between the sides also stayed balanced. It was concluded that occupational stress is present in this sample, but it does not correlate with the clinical findings of TMD and the electromyography examination of masticatory muscles in military personnel from the 5th CSM of the city of Ribeirão Preto/Brazil.
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The Assessment of Functional Abilities in the Diagnosis of MCI and Dementia in a Culturally Diverse SampleUnknown Date (has links)
Previous studies suggested that the Functional Activities Questionnaire (FAQ-10)
has minimal ethnic bias and that a shorter version (FAQ-6) can equally diagnose MCI
and dementia. Objective: We analyzed whether FAQ-6 is similar to FAQ-10 in
diagnosing MCI and dementia. We examined their applicability across European
Americans (EA) and Hispanic Americans, and how scores correlated to beta amyloid.
Method: 222 participants (116 EA) completed a neuropsychological battery, FAQ, and
PET scans, and were classified as cognitively normal (CN), MCI, or dementia. The
diagnostic capacity of FAQ-10 and FAQ-6 were compared for the total sample and across
ethnic groups. Scores were correlated to beta amyloid. Results: Both versions showed
good item discrimination. Ethnicity did not affect scores when controlling for diagnosis
and education. Both versions classified CN and dementia, and positively correlated to
beta amyloid. Conclusions: Results suggest FAQ-6 and FAQ-10 similarly predict
diagnosis and is adequate in these ethnic groups. / Includes bibliography. / Thesis (M.A.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection
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Multiple roles for the extracelllular matrix protein Tenascin-X in nerve gut functionAktar, Rubina January 2016 (has links)
Tenascin X (TNX) is a matricellular protein involved in regulating cellular functions by interacting with other extracellular matrix (ECM) proteins within the cell matrix and has anti-adhesive properties evidenced in tumours and wound healing. TNX is the only member of the tenascin family that is lost in Joint Hypermobility Syndrome (JHS) and exerts a crucial architectural function. Of importance, TNX deficient and JHS patients have gastrointestinal (GI) dysfunction. Despite this association no study has described the role of TNX in the GI tract. Thus, the aim of this thesis was to characterise the expression of TNX in the stomach and colon in mouse and human tissue. Second, we aimed to elucidate the functional role of TNX using TNX knockout (TNX KO) mice. Expression studies revealed TNX in vagal afferent endings in the mouse, and myenteric cell bodies in human stomach. In colon, TNX strongly associated with cholinergic submucous and myenteric neurons in both species, however, was not found in CGRP positive fibres. Cell bodies in nodose ganglia, dorsal root ganglia, ventral and dorsal horn were also TNX positive. Functional studies in stomach, using single fibre electrophysiology showed TNX KO mice had increased vagal afferent mechanoreceptor sensitivity. Octanoic acid breath test revealed rapid gastric emptying in TNX KO. Colonic manometry showed the amplitude and frequency of colonic contractions were reduced in TNX KO mice, particularly in the distal colon. Ussing chamber studies measuring changes in ion flux (indirect measure of secretion) showed no major difference between TNX KO and wild type (WT) mice. The specific localisation of TNX with neuronal structures in the gut is shown here for the first time suggesting that TNX is more than just an architectural protein. Indeed, its role in specific GI functions supports this observation and provides a mechanism for GI symptoms in JHS.
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A mechanistic investigation into candidate markers of telomere-induced senescence in normal human epidermal keratinocytesdos Santos Soares Martins de Castro, Alicia Maria January 2014 (has links)
Telomere dysfunction is one mechanism of cellular and tissue ageing. Dysfunctional telomeres in fibroblasts are recognised as DNA double-strand breaks (DSBs) and trigger the DNA damage pathway of senescence. However, telomere uncapping in normal human epidermal keratinocytes, via expression of the dominant negative mutant of the telomere repeat-binding factor 2 (TRF2!B!M), resulted in a senescent-like arrest without a significant DNA damage response (DDR). This suggests that either keratinocytes are unusually sensitive to telomere uncapping and the low DDR is sufficient to induce senescence or that dysfunctional telomeres may also be signalled through an alternative pathway. Subsequent analysis revealed genes HIST2H2BE, ICEBERG, S100A7 and HOPX as potential markers for telomere dysfunction-induced senescence (TDIS) since they were induced by telomere uncapping and seemed to be regulated by telomerase. The aim of this project was to assess the specificity of these candidate markers for TDIS and to select the most promising for use as a biomarker. To this end, keratinocytes were exposed to doses of ionising radiation, capable of generating transient or permanent damage to the DNA, or transduced with retroviral constructs expressing p14ARF, p16INK4a, p53 or TRF2!B!M and the gene expression levels of the candidates assessed after a recovery period or at the early stages of senescence. Whilst S100A7, HOPX or ICEBERG were not induced by a transient or persistent DDR or by p16INK4a, ICEBERG and HOPX were induced by p53 and p14ARF when these were ectopically expressed at higher levels. Thus, S100A7 seems to be the most specific early marker for telomere dysfunction in keratinocytes since it was selectively induced by telomere uncapping via expression of TRF2!B!M and not by DSBs or by over expression of p14ARF, p53 or p16INK4a. S100A7 may have the potential to identify cells with telomere dysfunction in human epithelia and body fluids.
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Effect of Diastolic Dysfunction on Postoperative Outcomes after Cardiovascular Surgery: a Systematic Review and Meta-AnalysisKaw, Roop, Hernandez, Adrian V., Pasupuleti, Vinay, Deshpande, Abhishek, Nagarajan, Vijaiganesh, Bueno, Hector, Coleman, Craig I., Ioannidis, John P.A., Bhatt, Deepak L., Blackstone, Eugene H. 06 1900 (has links)
El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objective The objective of this study was to investigate the effect of preoperative diastolic dysfunction on postoperative mortality and morbidity after cardiovascular surgery. Methods We systematically searched for articles that assessed the prognostic role of diastolic dysfunction on cardiovascular surgery in PubMed, Cochrane Library, Web of Science, Embase and Scopus until February 2016. Twelve studies (n=8224) met our inclusion criteria. Due to scarcity of outcome events, fixed-effects meta-analysis was performed using the Mantel-Haenszel method. Results Preoperative diagnosis of diastolic dysfunction was associated with higher postoperative mortality (OR 2.41, 95% CI 1.54-3.71; p<0.0001), major adverse cardiac events (MACE) (OR 2.07, 95% CI 1.55-2.78; p <=0.0001) and prolonged mechanical ventilation (OR 2.08, 95% CI 1.04-4.16; p=0.04) in comparison to patients without diastolic dysfunction among patients who underwent cardiovascular surgery. The odds of postoperative myocardial infarction (OR: 1.29, 95% CI 0.82, 2.05; p=0.28) and atrial fibrillation (OR: 2.67; 95% CI 0.49-14.43; p=0.25) did not significantly differ between the two groups. Severity of preoperative diastolic dysfunction was associated with increased postoperative mortality (OR 21.22, 95% CI 3.74 -120.33; p=0.0006) for Grade 3 diastolic dysfunction compared with patients with normal diastolic function. Inclusion of left ventricular ejection fraction (LVEF) <40% accompanying diastolic dysfunction, did not further impact postoperative mortality (p=0.27; I2 =18%) when compared with patients with normal LVEF and diastolic dysfunction. Conclusions Presence of preoperative diastolic dysfunction was associated with higher postoperative mortality and MACE, regardless of LVEF. Mortality was significantly higher in grade III diastolic dysfunction. Keywords Diastolic dysfunction; cardiovascular surgical procedures; mortality; meta-analysis / Revisión por pares
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Discursive power games in counselling psychologists' therapeutic accounts of working with male sexual dysfunction : a Foucauldian analysisJones, Lee January 2017 (has links)
Male sexual dysfunction is considered to be a problematic discursive site due to the diverse ways in which it is constructed and therapeutically conceptualised. Under-researched within the discipline of counselling psychology to date, this diagnostic category needs to be explored to identify ways in which counselling psychologists construct this presenting problem. Therefore the aim of this research was to interrogate how a volunteer group of counselling psychologists understood and worked with male sexual dysfunction in order to make visible some of the masked discursive practices related to its diverse constructions. Ten counselling psychologists were interviewed and a Foucauldian discourse analysis conducted, which interrogated the discursive power games implicated in these participants' accounts. The findings produced firstly identified the wider contextual cultural norms that seemed to regulate male sexuality within gendered masculinity discourses. Secondly, three distinct discursive therapeutic subject positions and their related power games were identified as talked about by these participants. Overall, it is argued that these findings indicate that for these counselling psychologists, male sexual dysfunction is a mutable, diversely power-laden, and thereby problematic, construct. Furthermore this analysis may be understood as a contribution to counselling psychology in raising practitioners' awareness to the power games in their talk about working with male sexual dysfunction.
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