Tyrosine 705 Phosphorylation of STAT3 Is Associated with Phenotype Severity in TGFβ1 Transgenic Mice

Transforming growth factor beta 1 (TGFβ1) is a key player in skeletal muscle degenerative and regenerative processes. We previously showed that conditionally overexpressing TGFβ1 in skeletal muscles caused myofiber atrophy and endomysial fibrosis in mice. However, the disease severity varied significantly among individual mice. While 40% of mice developed severe muscle pathology and lost body weight within 2 weeks of TGFβ1 transgene induction in muscles, the rest showed milder or no phenotype. This study aims at determining whether signal transducer and activator of transcription 3 (STAT3) plays a role in the phenotypic difference and whether it can be activated by TGFβ1 directly in muscle cells. Our results show that while total STAT3 was not differentially expressed between the two groups of mice, there was significantly higher pSTAT3 (Tyr705) in the muscles of the mice with severe phenotype. Immunohistochemistry showed that pSTAT3 (Tyr705) was localized in approximately 50% of the nuclei of the muscles. We further showed that TGFβ1 induced Tyr705 phosphorylation of STAT3 in C2C12 cells within 30 minutes of treatment while total STAT3 was not affected. Our findings suggest that TGFβ1 alone can induce Tyr705 phosphorylation of STAT3 in skeletal muscle cells and contribute to disease severity in transgenic TGFβ1 mice

Tyrosine 705 Phosphorylation of STAT3 Is Associated with Phenotype Severity in TGF β

Transforming growth factor beta 1 (TGFβ1) is a key player in skeletal muscle degenerative and regenerative processes. We previously showed that conditionally overexpressing TGFβ1 in skeletal muscles caused myofiber atrophy and endomysial fibrosis in mice. However, the disease severity varied significantly among individual mice. While 40% of mice developed severe muscle pathology and lost body weight within 2 weeks of TGFβ1 transgene induction in muscles, the rest showed milder or no phenotype. This study aims at determining whether signal transducer and activator of transcription 3 (STAT3) plays a role in the phenotypic difference and whether it can be activated by TGFβ1 directly in muscle cells. Our results show that while total STAT3 was not differentially expressed between the two groups of mice, there was significantly higher pSTAT3 (Tyr705) in the muscles of the mice with severe phenotype. Immunohistochemistry showed that pSTAT3 (Tyr705) was localized in approximately 50% of the nuclei of the muscles. We further showed that TGFβ1 induced Tyr705 phosphorylation of STAT3 in C2C12 cells within 30 minutes of treatment while total STAT3 was not affected. Our findings suggest that TGFβ1 alone can induce Tyr705 phosphorylation of STAT3 in skeletal muscle cells and contribute to disease severity in transgenic TGFβ1 mice

Pathogenetic Mechanisms of Collagen Type VI-Related Muscular Dystrophies

The overall focus of thesis is determining key disease-driving molecular pathways involved in the progression of a specific subtype of neuromuscular diseases, the collagen type VI-related muscular dystrophies (COL6RD). These kinds of muscular disorder encompass a wide spectrum of diseases, ranging from the severe Ullrich muscular dystrophy (UCMD) to the mild Bethlem myopathy (BM), characterized by a plethora of clinical manifestations. Both UCMD and BM usually arise at birth or early childhood with hypotonia and progressive muscle weakness, joint contractures and hyperlaxity of the distal joints. In UCMD, ambulation is achieved with a delay of 2 years compared to healthy children, but it is eventually lost. In BM, ambulation is generally maintained and only few affected individuals require wheel chair. Genetically, UCMD and BM are caused by mutations in the genes encoding for the three α-chains of the collagen type VI acting in a recessive or dominant way. Morphologically, the muscle biopsies of UCMD and BM patients are characterized by variable degree of atrophy, fibrosis, and regeneration and degeneration of the muscle fibers. In the present work, we systematically reviewed and classified a cohort of 22 COL6RD patient muscle biopsies and 14 age-matched control muscle biopsies according to basic histological features, such as muscle fibers atrophy and fibrosis. This classification was achieved through an innovative automatic image analysis algorithm and generated three groups, COL6RD-1, COL6RD-2 and COL6RD-3, of increasing histological severity. Microarray-based gene expression profiling and RNA-Seq of the same cohort produced gene expression fingerprint that suggested a strong segregation of COL6RD muscle biopsies from the control muscle biopsies, but did not clearly correlated with the histological grouping established among the patients. In silico analysis of the gene expression results revealed TGFβ1 pathway as the major regulator of the COL6RD histological changes and, most likely, of the underlying disease pathophysiology. TGFβ1 involvement was further confirmed by upstream regulator analysis, validation of downstream gene targets by qRT-PCR, and protein targets by western blot and immunofluorescence. Moreover, we also found a downregulation of skeletal muscle specific genes. RNA-Seq data also highlighted a deeper level of regulation of the gene expression carried over by alternative splicing. We identified 43 genes having more than one splice isoform whose expression level was significantly different between COL6RD muscle biopsies and controls, with DECORIN being the one displaying the highest number of alternatively spliced isoforms. Overall, this study suggests that different histological stages of COL6-RD are characterized by the same pattern of gene expression, involving predominantly the upregulation of matricellular components, even when the muscle involvement is not evident yet, and downregulation of muscle specific genes. Our results also suggest that TGFβ1 acts as a major driver of the pathological changes at the skeletal muscle level. Moreover, muscle biopsies from COL6-RD patients are characterized by an increase in alternative spliced isoforms of extracellular matrix-related genes that might contribute to the pathogenesis of the disease.L’obiettivo di questa tesi è determinare i meccanismi molecolari coinvolti nella progressione di un sottogruppo specifico di malattie neuromuscolari: le distrofie muscolari congenite causate da deficit di collagene di tipo VI. Questo gruppo di distrofie comprende un ampio spettro di disordini caratterizzati da un ampio spettro di manifestazioni cliniche: dalla più grave distrofia muscolare congenita di Ulrich (UCMD) alla più moderata miopatia di Bethlem” (BM). Sia UCMD che BM si manifestano alla nascita o nel prima infanzia con ipotonia e debolezza muscolare progressiva, contrazioni delle articolazioni prossimale e iperelasticità delle articolazioni distali. Individui affetti da UCMD iniziano a deambulare con un ritardo di 2 anni rispetto ad individui sani, e la capacità deambulatoria viene poi persa nuovamente. Individui affetti da BM, invece, conservano la capacità di ambulare e solo pochi necessitano di sussidi. Dal punto di vista genetico, UCMD e BM sono causate da mutazioni che possono colpire uno o più geni codificanti le tre molecole del collagene di tipo VI. Tali mutazioni possono agire in modo dominante o recessivo. Morfologicamente le biopsie muscolari di pazienti affetti da UCMD e BM sono caratterizzate da atrofia muscolare, fibrosi, rigenerazione e degenerazione delle fibre muscolare, evidenziata dalla presenza di aree necrotiche. Nel presente lavoro, abbiamo effettuato un’analisi sistematica di 22 biopsie muscolari ottenute da pazienti affetti da COL6RD e 14 biopsie muscolari ottenuti da controlli sani di età corrispondente, che sono state suddivise in base al grado di atrofia muscolare e fibrosi. Questa classificazione è stata effettuata usando un algoritmo originale per l’analisi automatica di preparazioni istologiche, ed ha generato 3 gruppi, COL6RD-1, COL6RD-2 and COL6RD-3, di gravità istologica crescente. Le stesse biopsie sono state sottoposte a microarray e RNA-Seq ed hanno rivelato andamenti di espressione genica in forte correlazione con i gruppi definiti secondo secondo i preparati istologici, sottolineando la chiara divisione tra le biopsie muscolari di individui affetti da COL6RD e controlli. L’analisi in silico dell’espressione genica ha successivamente rivelato TGFβ1 come il maggior responsabile dei cambiamenti istologici in COL6RD e, verosimilmente, dei processi patofisiologici sottostanti. Il coinvolgimento di TGFB1 è stato ulteriormente confermato dall’ upstream regulator analysis, validazione di geni target downstream tramite qRT-PCR, e di proteine effettrici tramite western blot e immunofluorescenza. I dati ottenuti tramite RNA-Seq hanno rivelato un ulteriore livello di regolazione dell’espressione genica attuato tramite splicing alternativo. Abbiamo identificato 43 geni aventi più di una variante la cui espressione è significativamente diversa tra biopsie muscolari di COL6RD e controlli sani. Nello specifico, Decorin è il gene con il maggior numero di isoforme originate da splicing alternativo. Nel complesso, questo studio suggerisce che i diversi stadi istologici di COL6RD sono caratterizzati dallo stesso profilo di espressione genica, che coinvolge prevalentemente l’aumento di componenti della matrice extracellulare, anche in mancanza di evidenze istologiche di fibrosi, e la diminuzione di prodotti di geni muscolo-specifici. I nostri risultati suggeriscono anche che ci siano pathway molecolari TGFβ1-dipendenti che agiscono da principali regolatori dei cambiamenti patologici a livello del muscolo scheletrico. Inoltre, le biopsie muscolari provenienti da pazienti affetti da COL6RD sono caratterizzate da un aumento delle isocrone geniche generate da splicing alternativo, soprattutto per geni coinvolti nella produzione e nel mantenimento della matrice extracellulare

STAT3 in Skeletal Muscle Function and Disorders

Signal transducer and activator of transcription 3 (STAT3) signaling plays critical roles in regulating skeletal muscle mass, repair, and diseases. In this review, we discuss the upstream activators of STAT3 in skeletal muscles, with a focus on interleukin 6 (IL6) and transforming growth factor beta 1 (TGF-β1). We will also discuss the double-edged effect of STAT3 activation in the muscles, including the role of STAT3 signaling in muscle hypertrophy induced by exercise training or muscle wasting in cachectic diseases and muscular dystrophies. STAT3 is a critical regulator of satellite cell self-renewal after muscle injury. STAT3 knock out affects satellite cell myogenic progression by impairing proliferation and inducing premature differentiation. Recent studies in STAT3 signaling demonstrated its direct role in controlling myogenic capacity of myoblasts and satellite cells, as well as the potential benefit in using STAT3 inhibitors to treat muscle diseases. However, prolonged STAT3 activation in muscles has been shown to be responsible for muscle wasting by activating protein degradation pathways. It is important to balance the extent of STAT3 activation and the duration and location (cell types) of the STAT3 signaling when developing therapeutic interventions. STAT3 signaling in other tissues and organs that can directly or indirectly affects skeletal muscle health are also discussed

Minimal Residual Disease assessment based on IgH rearrangements and flow cytometry in canine Diffuse Large B-cell Lymphoma

Introduction Canine diffuse large B-cell lymphoma (cDLBCL) accounts for 30-40% of lymphoma cases. The persistence of cells unidentifiable through cytology is termed Minimal Residual Disease (MRD). Currently, MRD is measured by flow-cytometry (FC). The aim of the study was to determine the diagnostic and prognostic role of rearranged IgH (PARR analysis) in the lymph node (LN), peripheral blood (PB) and bone marrow (BM) in cDLBCL compared with FC. Materials and methods PB, BM aspirate and LN tissue from 17 DLBCL were available for PARR analysis at diagnosis (T0) and at the end of treatment (T1). The concordance between PARR and FC was analyzed for LN, PB and BM. Results At T0, the B-cell origin of the lymphomas was confirmed by IgH Major rearrangements in the LN of all dogs. Eleven dogs (64.7%) showed simultaneous IgH rearrangements in BM and PB, whereas 6 dogs (35.3%) had no rearrangements in none of the samples. At T1, IgH monoclonal rearrangement was found in 13 (81%), 8 (50%) and 6 dogs (38%) in LN, PB and BM, respectively. At T0, the concordance rate between FC and PARR was 100%, 82% and 47% for LN, PB and BM, respectively. The highest concordance rate was obtained in LNs (PARR-positive/FC-positive), whereas the highest discordance was obtained in BM (PARR-positive/FC-negative). At T1, the concordance rate was overall reduced, accounting for 47%, 50%, and 63% in LN, PB and BM, respectively. The highest discordance rate was found in LNs (PARR-positive/FC-negative), whereas the highest concordance was found in BM (PARR-negative/FC-negative). Discussion We propose the use of both, PARR and FC, in tandem to offset the possibility of false-negative MRD assessment; in case of discrepant results, dogs should be closely monitored to detect a possible early recurrence. The concordance rate was largely dependent on the time point, being higher in T0. The discordance between PARR and FC results in dogs in clinical remission may be due to the limited number of available cells, which reduces the sensitivity of FC

Minimal residual disease detection by flow cytometry and PARR in lymph node, peripheral blood and bone marrow, following treatment of dogs with diffuse large B-cell lymphoma

The most promising techniques for detecting minimal residual disease (MRD) in canine lymphoma are flow cytometry (FC) and polymerase chain reaction amplification of antigen receptor genes (PARR). However, the agreement between these methods has not been established. MRD was monitored by FC and PARR following treatment of dogs affected with diffuse large B-cell lymphoma (DLBCL), comparing results in lymph node (LN), peripheral blood (PB) and bone marrow (BM) samples. The prognostic impact of MRD on time to relapse (TTR) and lymphoma-specific survival (LSS) was also assessed. Fourteen dogs with previously untreated DLBCL were enrolled into the study; 10 dogs eventually relapsed, while four dogs with undetectable MRD were still in remission at the end of the study. At diagnosis, the concordance rate between FC and PARR was 100%, 78.6%, and 64.3% for LN, PB and BM, respectively. At the end of treatment, the agreement rates were 35.7%, 50%, and 57.1% for LN, PB and BM, respectively. At least one of the follow-up samples from dogs experiencing relapse was PARR(+); conversely, FC was not able to detect MRD in seven of the dogs that relapsed. PARR was more sensitive than FC in predicting TTR, whereas the combination of PARR and FC was more sensitive than either technique alone in predicting LSS using PB samples. The results suggest that immunological and molecular techniques should be used in combination when monitoring for MRD in canine DLBCL

Hydrodynamic separation water flow sediment

The dissertation deals with clogging of reservoirs and ponds by redundant sediments which penetrate water system by erosion. It describes methods of extracting pond sediments. It contains measurement of dewatering of sediments on hydrocyclone and design of laboratory hydrocyclone and filter unit. In the conclusion there is design of technology of dewatering sediments which were extracted by wet method by drainage excavator. Numerical simulation of flowing in hydrocyclones is simulated in the CFX programme and dewatering technology is simulated in the CHEMCAD programme

Mitochondrial fission and remodelling contributes to muscle atrophy

Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin–proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme