Ibtissam Echchgadda

Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

Terahertz Radiation: A Non-contact Tool for the Selective Stimulation of Biological Responses in Human Cells

Collective motions of water and of many biological macromolecules have characteristic time scales on the order of a picosecond. As a result, these biomolecules can strongly absorb terahertz (THz) radiation. Due to this absorption, THz radiation can exert a diverse range of effects on biological structures. For example, THz radiation has been shown to impact the structure, functional activity, and dynamics of macromolecules such as DNA and proteins. THz radiation can affect several gene expression pathways and, consequently, can alter various biochemical and physiological processes in cells. Indeed, THz radiation has been shown to influence the expression of several genes within different cell types. However, a complete view of the global transcriptional responses and the intracellular canonical pathways specifically triggered by THz radiation has not been elucidated. In this study, we performed a global profiling of transcripts in human cells exposed to 2.52 THz radiation and compared the exposure responses to a thermally-matched bulk-heating (BH) protocol. Our results show that both THz radiation and BH induce a significant change in the expression of numerous mRNAs and microRNAs. The data also show that THz radiation triggers specific intracellular canonical pathways that are not affected in the BH-exposed cells. This study implies that THz radiation may be a useful, non-contact tool for the selective control of specific genes and cellular processes.

Using a portable terahertz spectrometer to measure the optical properties of in vivo human skin

Terahertz time-domain spectroscopy (THz-TDS) systems are capable of detecting small differences in water concentration levels in biological tissues. This feature makes THz devices excellent tools for the noninvasive assessment of skin; however, most conventional systems prove too cumbersome for limited-space environments. We previously demonstrated that a portable, compact THz spectrometer permitted measurement of porcine skin optical properties that were comparable to those collected with conventional systems. In order to move toward human use of this system, the goal for this study was to collect the optical properties, specifically the absorption coefficient (μa) and index of refraction (n), of human subjects in vivo. Spectra were collected from 0.1-2 THz, and measurements were made on the palm, ventral (inner) and dorsal (outer) forearm. Prior to each THz measurement, we used a multiprobe adapter system to measure each subject’s skin hydration levels, transepidermal waterloss (TEWL), skin color, and degree of melanin pigmentation. Our results suggest that the measured optical properties were wide-ranging, and varied considerably for skin tissues with different hydration and melanin levels. These data provide a novel framework for accurate human tissue measurements using THz spectrometers in limited-space environments.

Terahertz spectroscopy of dry, hydrated, and thermally denatured biological macromolecules

Terahertz time-domain spectroscopy (THz-TDS) is an effective technique to probe the intermolecular and collective vibrational modes of biological macromolecules at THz frequencies. To date, the vast majority of spectroscopic studies have been performed on dehydrated biomolecular samples. Given the fact that all biochemical processes occur in aqueous environments and water is required for proper protein folding and function, we hypothesize that valuable information can be gained from spectroscopic studies performed on hydrated biomolecules in their native conformation. In this study, we used a THz-TDS system that exploits photoconductive techniques for THz pulse generation and freespace electro-optical sampling approaches for detection. We used the THz spectrometer to measure the time-dependent electric field of THz waves upon interaction with water, phosphate buffered saline (PBS), and collagen gels. By comparing these waveforms with references, we simultaneously determined each samples index of refraction (n) and absorption coefficients (μa) as a function of frequency. Our data show that the properties we measure for the water, PBS and collagen are comparable to those reported in the literature. In the future, we plan to examine the effect that both temperature and pH have on the optical properties of other biological macromolecules. Studies will also be performed to compare our results to those generated using molecular dynamics simulations.

Qualitative Behavior of the Low-Frequency Vibrational Dynamics of Microtubules and the Surrounding Water

The dynamics of the low-frequency vibrational modes of microtubules play a key role in many theoretical models regarding their biological function. We analyze these dynamics through large scale, classical molecular dynamics simulations of a microtubule composed of 42 tubulin heterodimers to provide insights into the qualitative nature of the vibrational energy absorption and dissipation mechanisms. The computed microtubule absorption spectra and vibrational density of states in the terahertz regime are presented, along with an analysis of the vibrational dephasing rates of the tubulin monomer center of mass dynamics, which are shown to be overdamped. Additionally, the presence of the microtubule modifies the dynamical properties of the solvation shell structure within roughly 10 Å of the protein. These vibrational properties are similar to those seen in other globular proteins and indicate microtubules are unlikely candidates for any large scale collective vibrational processes in the terahertz regime such as Fröhlich condensates.

Determination of the optical properties of melanin-pigmented human skin equivalents using terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) methods have been utilized in previous studies in order to characterize the optical properties of skin and its primary constituents (i.e., water, collagen, and keratin). However, similar experiments have not yet been performed to investigate whether melanocytes and the melanin pigment that they synthesize contribute to skin’s optical properties. In this study, we used THz-TDS methods operating in transmission geometry to measure the optical properties of in vitro human skin equivalents with or without normal human melanocytes. Skin equivalents were cultured for three weeks to promote gradual melanogenesis, and THz time domain data were collected at various time intervals. Frequency-domain analysis techniques were performed to determine the index of refraction (n) and absorption coefficient (μa) for each skin sample over the frequency range of 0.1-2.0 THz. We found that for all samples as frequency increased, n decreased exponentially and the μa increased linearly. Additionally, we observed that skin samples with higher levels of melanin exhibited greater n and μa values than the non-pigmented samples. Our results indicate that melanocytes and the degree of melanin pigmentation contribute in an appreciable manner to the skin’s optical properties. Future studies will be performed to examine whether these contributions are observed in human skin in vivo.

Temporal Gene Expression Kinetics for Human Keratinocytes Exposed to Hyperthermic Stress

The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the “recovery phase”, roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress.

Adult human dermal fibroblasts exposed to nanosecond electrical pulses exhibit genetic biomarkers of mechanical stress

Background Exposure of cells to very short (<1 µs) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulses (nsEP) can cause disruption of the plasma membrane, cellular swelling, shrinking and blebbing. Molecularly, nsEP have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. We hypothesize that studying the genetic response of primary human dermal fibroblasts exposed to nsEP, will gain insight into the molecular mechanism(s) either activated directly by nsEP, or indirectly through electrophysiology interactions. Methods Microarray analysis in conjunction with quantitative real time polymerase chain reaction (qRT-PCR) was used to screen and validate genes selectively upregulated in response to nsEP exposure. Results Expression profiles of 486 genes were found to be significantly changed by nsEP exposure. 50% of the top 20 responding genes coded for proteins located in two distinct cellular locations, the plasma membrane and the nucleus. Further analysis of five of the top 20 upregulated genes indicated that the HDFa cells’ response to nsEP exposure included many elements of a mechanical stress response. Conclusions We found that several genes, some of which are mechanosensitive, were selectively upregulated due to nsEP exposure. This genetic response appears to be a primary response to the stimuli and not a secondary response to cellular swelling. General significance This work provides strong evidence that cells exposed to nsEP interpret the insult as a mechanical stress.

Effects of different terahertz frequencies on gene expression in human keratinocytes

In recent years, a surge in the development of many terahertz (THz) sensing and imaging technologies occurred leading to increased use in military and civil operations. Therefore, understanding the biological effects associated with exposures to this radiation is becoming increasingly important. Previous studies have speculated that cells exposed to different frequencies of THz radiation may exhibit differential responses. However, empirical studies to confirm such differences have not been performed. The question of whether cells exposed to different THz frequencies exhibited specific biological responses remains unclear. In this study, we exposed human keratinocytes to a THz laser tuned to several different THz frequencies using our recently developed THz exposure system. This system consists of an optically pumped molecular gas THz laser source coupled to a modified cell culture incubator permitting THz radiation exposures under controlled standard tissue culture conditions. For all frequencies, we matched the THz exposure duration and irradiance. During THz exposure, we monitored the power as DC voltage-logged values (LabVIEW™ IV log). To determine the temperature changes by THz exposure, we collected temperature readings from the unexposed and THz-exposed cells using thermocouples. We assessed cellular viability after exposure using MTT colorimetric assays. We compared the changes in gene expression profiles using messenger RNA (mRNA) microarrays, and we identified the THz-induced signaling pathways for each frequency using bioinformatics. Our data provide valuable new insights that give a comparative picture of the genes and intracellular signaling pathways triggered in cells exposed to THz radiation at different frequencies.

State-of-the-art exposure chamber for highly controlled and reproducible THz biological effects studies

Terahertz (THz) imaging and sensing technologies are increasingly being used at international airports for security screening purposes and at major medical centers for cancer and burn diagnosis. The emergence of new THz applications has directly resulted in an increased interest regarding the biological effects associated with this frequency range. Knowledge of THz biological effects is also desired for the safe use of THz systems, identification of health hazards, and development of empirically-based safety standards. In this study, we developed a state-of-the-art exposure chamber that allowed for highly controlled and reproducible studies of THz biological effects. This innovative system incorporated an industry grade cell incubator system that permitted a highly controlled exposure environment, where temperatures could be maintained at 37 °C ± 0.1 °C, carbon dioxide (CO2) levels at 5% ± 0.1%, and relative humidity (RH) levels at 95% ± 1%. To maximize the THz power transmitted to the cell culture region inside the humid incubator, a secondary custom micro-chamber was fabricated and incorporated into the system. This micro-chamber shields the THz beam from the incubator environment and could be nitrogen-purged to eliminate water absorption effects. Additionally, a microscope that allowed for real-time visualization of the live cells before, during, and after THz exposure was integrated into the exposure system.