PLATFORM FOR CALCIUM IMAGING, ISOLATION, AND CHARACTERIZATION IN PRIMARY COLON EPITHELIAL CELLS

The colonic epithelium, lining the innermost portion of the large intestine, is subjected to continued mechanical and chemical stress therefore requiring constant self-renewal. This renewal is supplied from the colon crypts which maintain and protect the intestinal stem cells in an undifferentiated state at the base. Through a multitude of growth factors, metabolites, gases, and extracellular matrix gradients along the colon crypt contributing to proliferation at the base and differentiation as cells migrate upwards. Recently, Ca2+ signaling has been identified as an integrator of multiple mitogenic signaling contributing to the regulation of stem-cell differentiation. These findings have yet to be fully explored with primary mammalian colon cells and face challenges in imaging 3D architecture such as organoids. Chapter 2 addresses these challenges performing calcium imaging in 2D primary murine colon monolayers. Adopting imaging strategies originally developed for neuron calcium imaging, and image analysis pipeline was built capable of accurately isolating and extracting Ca2+ cell signals from the colon epithelium. Analyzing the isolate Ca2+ showed a correlation between higher frequencies and lower pulse width with proliferative cells, while broader oscillations occurring at a lower frequency were associated with differentiation. In Chapter 3 the impact of different growth factors is shown on the spatial temporal properties of the colon cell Ca2+ signals, demonstrating a similar relationship between higher frequency Ca2+ signals and proliferation. Chapter 4 improves on the improves on the previous work introducing cells zones undergoing proliferation and differentiation, allowing for more targeted investigation in the Ca2+ signaling found in proliferative colon cells.Doctor of Philosoph

Overexpression of HSP70 in mouse skeletal muscle protects against muscle damage and age‐related muscle dysfunction

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154339/1/fsb2fj030395fje-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154339/2/fsb2fj030395fje.pd

Effect of lifelong overexpression of HSP70 in skeletal muscle on age‐related oxidative stress and adaptation after nondamaging contractile activity

[EN] Skeletal muscle aging is characterized by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe injury. The ability of muscles of old mice to produce heat shock proteins (HSPs) in response to stress is severely diminished. Studies in our laboratory using HSP70 overexpressor mice demonstrated that lifelong overexpression of HSP70 in skeletal muscle provided protection against damage and facilitated successful recovery after damage in muscles of old mice. The mechanisms by which HSP70 provides this protection are unclear. Aging is associated with the accumulation of oxidation products, and it has been proposed that this may play a major role in age-related muscle dysfunction. Muscles of old wild-type (WT) mice demonstrated increased lipid peroxidation, decreased glutathione content, increased catalase and superoxide dismutase (SOD) activities, and an inability to activate nuclear factor (NF)- B after contractions in comparison with adult WT mice. In contrast, levels of lipid peroxidation, glutathione content, and the activities of catalase and SOD in muscles of old HSP70 overexpressor mice were similar to adult mice and these muscles also maintained the ability to activate NF- B after contractions. These data provide an explanation for the preservation of muscle function in old HSP70 overexpressor mice.—Broome, C. S., Kayani, A. C., Palomero, J., Dillmann, W. H., Mestril, R., Jackson, M. J., McArdle, A. Effect of lifelong overexpression of HSP70 in skeletal muscle on age-related oxidative stress and adaptation after nondamaging contractile activity

Minimal access surgery in emergency surgical conditions, impact beyond the aesthetic

En el año 2016, el Servicio de Cirugía General realizó 4432 intervenciones quirúrgicas, de ellas el 45 % se realizó de forma urgente. Con el objetivo de mostrar el impacto que ha generado la aplicación de la cirugía de mínimo acceso en las afecciones quirúrgicas de urgencia en el orden científico, social y económico, se realizó una investigación básica, descriptiva y transversal en 187 pacientes intervenidos por cirugía de mínimo acceso de urgencia, en el Servicio de Cirugía General del Hospital Provincial Clínico Quirúrgico Docente Celia Sánchez Manduley de Manzanillo, desde enero 2012 hasta diciembre 2016. Las principales variables se compararon con una muestra similar de pacientes intervenidos por vía abierta. La apendicectomía y la colecistectomía fueron las intervenciones más representativas con un 33,15 % y 29,41 % respectivamente, aunque se realizaron lavados peritoneales e intervenciones sobre el tórax. La media del tiempo quirúrgico no sobrepasó los 50 minutos para toda la serie. El 9,6 % de las operaciones fueron reintervenciones, con un 94,4 % de evolución favorable. El 3,74 % de las intervenciones fueron convertidas con solo 5 complicaciones. El 9,6 % de las operaciones fueron reintervenciones. Se produjo un ahorro cuantioso derivado de la reducción de gastos hospitalarios e incorporación precoz a las actividades sociales y productivas. Las pocas molestias perioperatorias y la ambulatorización de la atención médica permitieron escasos desajustes familiares y mayor satisfacción para el paciente y la sociedad.In 2016, the General Surgery Service performed 4432 surgical procedures, of which 45 % were performed urgently. In order to show the impact generated by the application of the minimally invasive surgery to emergency surgical conditions in the scientific, social and economic, a basic, descriptive and cross-sectional research was carried out in 187 patients operated by emergency minimally invasive surgery, in the General Surgery Service of the Celia Sánchez Manduley Provincial Teaching Surgical Clinic Hospital of Manzanillo, from January 2012 to December 2016. The main variables were compared with a similar sample of patients who underwent open surgery. Appendectomy and cholecystectomy were the most frequent interventions representative with 33.15 % and 29.41 %; respectively, although peritoneal lavages and interventions on the thorax were performed. The average time surgery did not exceed 50 minutes for the entire series 9.6 % of the operations were reinterventions with a 94.4 % favorable evolution, 3.74 % of the interventions were converted with only 5 complications, 9.6 % of the operations were reinterventions. There was a substantial saving derived from the reduction of hospital expenses and early incorporation into social and productive activities. The few perioperative discomforts and the ambulatoryization of medical care allowed few family imbalances and greater satisfaction for the patient and society

Spontaneous neural synchrony links intrinsic spinal sensory and motor networks during unconsciousness

Non-random functional connectivity during unconsciousness is a defining feature of supraspinal networks. However, its generalizability to intrinsic spinal networks remains incompletely understood. Previously, Barry et al., 2014 used fMRI to reveal bilateral resting state functional connectivity within sensory-dominant and, separately, motor-dominant regions of the spinal cord. Here, we record spike trains from large populations of spinal interneurons in vivo in rats and demonstrate that spontaneous functional connectivity also links sensory- and motor-dominant regions during unconsciousness. The spatiotemporal patterns of connectivity could not be explained by latent afferent activity or by populations of interconnected neurons spiking randomly. We also document connection latencies compatible with mono- and disynaptic interactions and putative excitatory and inhibitory connections. The observed activity is consistent with the hypothesis that salient, experience-dependent patterns of neural transmission introduced during behavior or by injury/disease are reactivated during unconsciousness. Such a spinal replay mechanism could shape circuit-level connectivity and ultimately behavior

The importance of the cellular stress response in the pathogenesis and treatment of type 2 diabetes

Organisms have evolved to survive rigorous environments and are not prepared to thrive in a world of caloric excess and sedentary behavior. A realization that physical exercise (or lack of it) plays a pivotal role in both the pathogenesis and therapy of type 2 diabetes mellitus (t2DM) has led to the provocative concept of therapeutic exercise mimetics. A decade ago, we attempted to simulate the beneficial effects of exercise by treating t2DM patients with 3 weeks of daily hyperthermia, induced by hot tub immersion. The short-term intervention had remarkable success, with a 1 % drop in HbA1, a trend toward weight loss, and improvement in diabetic neuropathic symptoms. An explanation for the beneficial effects of exercise and hyperthermia centers upon their ability to induce the cellular stress response (the heat shock response) and restore cellular homeostasis. Impaired stress response precedes major metabolic defects associated with t2DM and may be a near seminal event in the pathogenesis of the disease, tipping the balance from health into disease. Heat shock protein inducers share metabolic pathways associated with exercise with activation of AMPK, PGC1-a, and sirtuins. Diabetic therapies that induce the stress response, whether via heat, bioactive compounds, or genetic manipulation, improve or prevent all of the morbidities and comorbidities associated with the disease. The agents reduce insulin resistance, inflammatory cytokines, visceral adiposity, and body weight while increasing mitochondrial activity, normalizing membrane structure and lipid composition, and preserving organ function. Therapies restoring the stress response can re-tip the balance from disease into health and address the multifaceted defects associated with the disease

A microarray study of MPP(+)-treated PC12 Cells: Mechanisms of toxicity (MOT) analysis using bioinformatics tools

BACKGROUND: This paper describes a microarray study including data quality control, data analysis and the analysis of the mechanism of toxicity (MOT) induced by 1-methyl-4-phenylpyridinium (MPP(+)) in a rat adrenal pheochromocytoma cell line (PC12 cells) using bioinformatics tools. MPP(+ )depletes dopamine content and elicits cell death in PC12 cells. However, the mechanism of MPP(+)-induced neurotoxicity is still unclear. RESULTS: In this study, Agilent rat oligo 22K microarrays were used to examine alterations in gene expression of PC12 cells after 500 μM MPP(+ )treatment. Relative gene expression of control and treated cells represented by spot intensities on the array chips was analyzed using bioinformatics tools. Raw data from each array were input into the NCTR ArrayTrack database, and normalized using a Lowess normalization method. Data quality was monitored in ArrayTrack. The means of the averaged log ratio of the paired samples were used to identify the fold changes of gene expression in PC12 cells after MPP(+ )treatment. Our data showed that 106 genes and ESTs (Expressed Sequence Tags) were changed 2-fold and above with MPP(+ )treatment; among these, 75 genes had gene symbols and 59 genes had known functions according to the Agilent gene Refguide and ArrayTrack-linked gene library. The mechanism of MPP(+)-induced toxicity in PC12 cells was analyzed based on their genes functions, biological process, pathways and previous published literatures. CONCLUSION: Multiple pathways were suggested to be involved in the mechanism of MPP(+)-induced toxicity, including oxidative stress, DNA and protein damage, cell cycling arrest, and apoptosis

Phylogeny Disambiguates the Evolution of Heat-Shock cis-Regulatory Elements in Drosophila

Heat-shock genes have a well-studied control mechanism for their expression that is mediated through cis-regulatory motifs known as heat-shock elements (HSEs). The evolution of important features of this control mechanism has not been investigated in detail, however. Here we exploit the genome sequencing of multiple Drosophila species, combined with a wealth of available information on the structure and function of HSEs in D. melanogaster, to undertake this investigation. We find that in single-copy heat shock genes, entire HSEs have evolved or disappeared 14 times, and the phylogenetic approach bounds the timing and direction of these evolutionary events in relation to speciation. In contrast, in the multi-copy gene Hsp70, the number of HSEs is nearly constant across species. HSEs evolve in size, position, and sequence within heat-shock promoters. In turn, functional significance of certain features is implicated by preservation despite this evolutionary change; these features include tail-to-tail arrangements of HSEs, gapped HSEs, and the presence or absence of entire HSEs. The variation among Drosophila species indicates that the cis-regulatory encoding of responsiveness to heat and other stresses is diverse. The broad dimensions of variation uncovered are particularly important as they suggest a substantial challenge for functional studies