Browsing by Subject "Temperature"

Chronic and acute back injuries are widespread, affecting people in environments where they are exposed to vibration and repeated shock. These issues have been widely reported among personnel on aircraft and small watercraft; operators of heavy industrial or construction equipment may also experience morbidity associated with cyclic loading. To prevent these types of injuries, an improved understanding is needed of the spine’s tolerance to fatigue injury and of the factors that affect fatigue tolerance.

These types of vibration and shock exposures are addressed by international standards that propose limitations on the length and severity of the accelerations to which an individual is subjected. However, the current standard, ISO 2631-5:2004, provides an imprecise health hazard assessment. In this dissertation, a detailed technical critique is presented to examine the assumptions on which ISO 2631-5:2004 is based. An original analysis of existing data yields an age-based regression of the ultimate strength of lumbar spinal units and demonstrates sources of error in the strength regression in the standard. This dissertation also demonstrates that, contradicting earlier assumptions, the ultimate strength of the spine does not lie on a power-law S-N curve, and fatigue tolerance cannot be extrapolated from ultimate strength tests.

An alternative approach is presented for estimating the injury risk due to repeated loading. Drawing from existing data in the literature, a large dataset of in vitro fatigue tests of lumbar spinal segments was assembled. Using this fatigue data, a survival analysis approach was used to estimate the risk of failure based on several factors. Number of cycles, load amplitude, sex, and age all were significant predictors of bony failure in the spinal column. The parameter described by ISO 2631-5:2004 to quantify repeated loading exposure was modified, and an injury risk model was developed based on this modified parameter which relates risk of vertebral failure to repeated compressive loading. Notably, the effect of sex on fatigue tolerance persisted after normalizing by area, emphasizing the need for men and women to be addressed separately in the creation of injury risk predictions and occupational guidelines.

Posture has also been implicated in altering the injury mechanisms and tolerance to fatigue loading. However, few previous investigations in cyclic loading have addressed non-neutral postures. To assess the influence of dynamic flexion on the fatigue tolerance of the lumbar spine, a series of tests were conducted which combined a cyclic compressive force with a dynamic flexing motion. A study of 17 spinal segments from six young male cadavers was conducted, with tests ranging from 1000 to 500 000 cycles. Of the 17 specimens, 7 failed during testing. These failures were analyzed using a Cox Proportional Hazards model. As in compressive fatigue behavior, significant factors were the magnitude of the applied load and the age of the specimen. However, when the dynamically flexed specimens in these tests were compared to the specimens in the axial fatigue dataset, the flexion condition did not have a detectable effect on fatigue tolerance.

The Hybrid III dummy is a critical tool the assessment of such loading. Although the Hybrid III was originally designed for automotive frontal impact testing, these dummies have since been used to measure exposures and estimate injury risks of a wide variety of scenarios. These scenarios often involve using the dummy under non-standard temperatures or with little recovery interval between tests. Series of tests were conducted on the Hybrid III neck and lumbar components to assess the effects of rest duration intervals and a range of temperatures. Variations in rest duration intervals had little effect on the response of either component. However, both components were extremely sensitive to changes in temperature. For the 50th percentile male HIII neck, the stiffness fell by 18% between 25°C and 37.5°C; at 0°C, the stiffness more than doubled, increasing by 115%. Temperature variation had an even more pronounced effect on the HIII lumbar. Compared to room temperature, the lumbar stiffness at 37.5°C fell by 40%, and at 12.5°C, the stiffness more than doubled, increasing by 115%.

This dissertation has advanced the state of knowledge about the fatigue characteristics of the spine. An injury risk function has been developed that can serve as a tool for health hazard assessment in occupational standards. It has also contributed a fatigue dataset with dynamic flexion. This work will improve the scientific community’s ability to prevent repeated loading injuries. This dissertation has also demonstrated the immense sensitivity to temperature of the Hybrid III spinal components. This finding has major implications for the interpretation of previously published work using the Hybrid III, for the conduct of future research, and for future dummy design.

Background

Methodology/principal findings

Conclusions

The vertebrate gonad is an excellent model to study organogenesis due to its unique ability to form two distinct organs from a common bipotential primordium. No single factor is responsible for activation of ovary or testis development in all vertebrate species, but these developmental pathways tend to converge on the same cohort of genetic regulators. The structures of testes and ovaries are extremely similar across vertebrates, and this high level of conservation is also observed in the gene regulatory processes underlying their differentiation. In heterogametic species such as mice and chickens, genes on the sex chromosomes activate the genes that drive differentiation of the testis of ovary. However, not all vertebrate species have sex chromosomes, and it’s unknown how the many genetic and cellular processes that direct gonad development are activated in the absence of a clear genetic signal. Temperature-dependent sex determination (TSD) is one of the primary sex determination strategies found in reptiles and has repeatedly evolved in multiple reptilian lineages. During TSD, the fate of the gonad is driven by nest temperatures experienced during embryonic development. In the decades since TSD was first described, the molecular processes underlying this phenomenon have remained a mystery.

The Red-Eared Slider turtle, Trachemys scripta elegans (T. scripta), is a widely-studied model for temperature-dependent sex determination. When eggs are incubated at a constant 26˚C, 100% of embryos will develop testes. Incubating eggs at a constant 31˚C produces only embryos with ovaries. Prior work has focused the regulation of aromatase, which is crucial to estrogen synthesis, but it is expressed relatively late in the sex determination window. A transcriptome analysis of T. scripta gonads through sex determination revealed a group of early, male-biased genes, including the H3K27 demethylase Kdm6b. In many vertebrates, the epigenetic state of key sex determining genes appears to be critical in the activation of testis or ovary specific-signaling. We investigated whether KDM6B mediates the effect of temperature on gene expression in T. scripta and we found that it activates a conserved regulator of male sex development, DMRT1.

One of the few identified transcriptional regulators of Kdm6b, the transcription factor STAT3, is only phosphorylated at the warmer, female-producing temperature (FPT). We show that pSTAT3 binds the Kdm6b locus to repress transcription and inhibition of pSTAT3 is sufficient to induce female-to-male sex reversal. Using primary cells derived from T. scripta gonads, we found that a heat-mediated influx of calcium at FPT promotes phosphorylation of STAT3. From these data we propose the model that heat-mediated influx of calcium at FPT promotes activation of STAT3, a transcriptional repressor of the male pathway. Our model is the first proposed mechanism of temperature-dependent sex determination supported by direct experimental evidence.

It is unknown how the gonad interprets environmental signals and coordinates cell fates across the tissue. The embryonic gonad coelomic epithelium is a common feature of many vertebrate gonads, and its development is critical to placement of the germ cells in the appropriate stem cell niche, which is required for germ cell survival and maturation. Previous studies of testis morphogenesis in T. scripta show that invaginations of the coelomic epithelium move germ cells into the gonad medulla to form the seminiferous tubules. We show that these invaginations only occur below germ cells, express the conserved Steroli cell marker SOX9, and are sensitive to the hormone environment of the gonad. These data suggest that signals between the germ cell, somatic cells in the coelomic epithelium, and somatic cells of the primordial cords collectively participate in the morphogenetic changes underlying testis development in T. scripta.

Our findings provide a framework for future investigations into the mechanism underlying temperature-dependent sex determination by identifying the initial signaling events that regulate the epigenetic state of sex-specific genes and describing how cellular fates are maintained during the sex determination window. STAT3 signaling can be activated by many inputs and have numerous downstream impacts, only some of which have been experimentally tested, providing direction and future lines of investigation for the field. The data presented here has laid the groundwork for identifying how temperature-sex determination operates in the turtle and how pieces of this process may be conserved among many animal phyla.