| dc.description.abstract | In recent years, biophotonics has become a rapidly emerging area in modern research with a great deal of promise for the future. Biophotonics allows the study of the interaction of light with biological species. It is an interdisciplinary branch of science covering wide areas of biology, medicine, physics, chemistry, mathematics, and engineering. Since long, efforts have been in progress to explore the interface of light and life (bio + photonics) to understand the structural and functional aspects of the smallest living systems of life like cells and more complex systems like tissues and organs. In light of the latest technological advancements, vibrational microspectroscopic (Infrared and Raman) techniques have emerged as important tools in the areas of biology and medicine. These techniques have enabled us to understand the chemical and physical nature of biological samples down to the molecular level. The sensitive, fast, and reproducible data produced by these spectroscopic techniques have made them effective and efficient tools available to scientists.
The present thesis has been divided into nine chapters and primarily deals with the application of IR microscopy to study human hair, brain cancer tissues, and cell-drug interactions. Chapter 1 gives a brief literature review of applications of infrared (IR) microspectroscopy in the field of biology and medicine. IR microscopy has an immediate appeal in biology and medicine because it is fast and non-invasive in nature. Photon energies in the IR region are too low to induce any kind of photochemical, photo-toxic, or photo-bleaching effects in biological samples. IR spectroscopy allows easy visualization of the cellular constituents and, based on their intrinsic chemical properties, provides a potential route to obtain diagnostic markers for diseases.
To probe the subtle changes occurring at the molecular level, IR microspectroscopy has been used as a non-destructive molecular descriptor. It is a non-invasive photonic technique to understand the intrinsic chemical composition of biological samples and modifications therein brought by abnormal conditions in the biological system. The technique is sensitive enough to detect finer variations occurring within biomolecules such as proteins, lipids, DNA, carbohydrates, etc., which contribute toward the formation of the sample of interest like cells and tissues. This technique not only minimizes extensive sample preparation by eliminating any requirement for contrast-inducing stains or molecular probes but is also capable of bringing out the inherent chemical heterogeneity as seen in the morphology of the sample. A remarkable advantage of this technique lies in the fact that each pixel in the imaging system corresponds to an IR spectrum. This reflects the chemical as well as spatial heterogeneity in tissue sections based on variations in the peak positions, shapes, and relative intensities.
Chapter 2 deals with the experimental methodologies and also describes some of the common experimental difficulties encountered during IR microscopy measurements. Causes responsible for these problems and necessary precautions have been discussed to avoid such problems.
Chapter 3 presents a model study to understand the effect of surfactants and dyes on the physicochemical properties of human hair. The results in the chapter have been discussed in two sections. In the first section, the chemical changes induced by sodium dodecyl sulfate (SDS), a well-known surfactant used in shampoo formulations, on human scalp hair have been discussed. In particular, FTIR ATR microscopy has been employed to understand the SDS-induced changes in the secondary structure of protein present in the outer protective layer of hair, i.e., the cuticle. Conformational changes in the secondary structure of protein were studied by curve fitting of the amide I band after every phase of SDS treatment. It has been found that SDS brings rearrangements in the protein backbone conformations by transforming ?-sheet structure to random coil and ?-turn. Additionally, AFM and SEM studies carried out to understand the morphological changes induced on the hair surface demonstrate the rupture and partial erosion of cuticle sub-layers.
The second section of the chapter focuses on understanding the effect of natural (Henna) and synthetic dyes on human hair using FTIR ATR microscopy. Experimental results suggest that subsequent to the use of Henna and synthetic dye, chemical and morphological properties of the hair fiber were altered. FTIR ATR experiments indicated that Henna and synthetic dye oxidize the disulfide bonds of hair proteins to cysteic acid. The damage caused by the synthetic dye was substantial in comparison to that induced by Henna, which is a natural dye. AFM experiments indicated that the topography and morphology of hair are altered as a consequence of hair coloration. Damage to the cuticle layer increased after every color treatment, resulting in cracks, holes, and partial erosion of cuticle sub-layers. The damage brought by synthetic dye in terms of morphological changes was considerable in comparison to that introduced by Henna. SEM studies were consistent with AFM results.
Chapter 4 deals with the IR study of glioblastoma (GBM, WHO grade IV brain cancer) and intermediate filament proteins. In this chapter, results have been discussed in two sections. In the first section, we have discussed the use of FTIR imaging to characterize and differentiate between normal and GBM tissue sections. GBMs are known to show extensive heterogeneity in tissue morphology, which directly corresponds to the biochemical microenvironment of the cells. In this chapter, cellular heterogeneity in GBM tissue sections has been characterized spectroscopically. The different cell types studied include fibrillary, pleomorphic, small, giant, and lipidized astrocytes. IR absorbance intensities (at various frequencies) were determined along with their band shapes. These values were used to distinguish various cell types within tissue sections.
Firstly, necrosis, which is a hallmark of GBM, was differentiated from cellular proliferation. Secondly, normal and GBM samples were distinguished, and thirdly, normal and different cell types within GBM were differentiated based on various spectral markers. Finally, different cell types within GBM were distinguished from one another to develop a robust classification method. The present study could help in automated spectroscopic identification of GBMs, especially in:
The diagnosis of stereotactic biopsies where small sample size often poses challenges.
Frozen sections which lose cellular morphology at low temperature.
Prognosis of GBMs, as some cell types are believed to have better survival benefits than others.
The robustness of the database built during this study was evaluated by the classification of unknown (blind) samples. The second section covers the study carried out to assign the 1338 cm?¹ IR band present in some of the GBM samples. This study establishes that the 1338 cm?¹ band is related to intermediate filament proteins (IFPs) present in the brain. IFPs are cytoskeletal proteins that play a pivotal role in maintaining the structure and functions of cells. These proteins are known to be responsible for the mechanical strength, shape, and rigidity of cells. In the present study, we have employed FTIR microscopy to probe IFPs present in the brain and have demonstrated that the IR band at 1338 cm?¹ corresponds to the broad family of IFPs, apart from collagen, which is an extracellular matrix protein. IR results have been further supported by immunohistochemical assay, which clearly indicates the association of IFPs present in the brain with the band at 1338 cm?¹. In addition, experiments on glioma cells (U343 and U373) and human scalp hair samples, which have minimal amounts of collagen, reconfirmed our assignment of the band at 1338 cm?¹.
Chapter 5 deals with the study of cell-drug interactions. In this chapter, we have employed IR microscopy to evaluate modifications in cellular macromolecules subsequent to treatment with various histone deacetylase inhibitors (HDIs). HDIs act against histone deacetylase (HDAC) enzymes, which have essential roles in gene regulation; thus, they are important target enzymes in anti-cancer drug research. Acetylation and deacetylation of nucleosomal histones are vital processes for proper cell function. In cells, reversible acetylation of histones is mediated by histone acetyltransferase (HAT) enzymes, which facilitate acetylation, whereas HDAC enzymes remove the acetyl groups. HDIs result in acetylation on lysine residues in the N-terminal of nucleosomal histones as a consequence of suppression of HDACs.
In addition to CH? (methyl) stretching bands at 2872 and 2960 cm?¹, which arise due to acetylation, major changes in the intensity of the bands at 2851 and 2922 cm?¹ have been observed, which originate from stretching vibrations of CH? (methylene) groups, in valproic acid-treated cells. Recently, HDIs have been shown to induce propionylation besides acetylation. Since propionylation involves CH? groups, we hypothesized that CH?-specific vibrational frequency changes observed in HDI-treated cells could be associated with propionylation. This was further confirmed by Western blot experiments using propionyl-lysine-specific antibody. Thus, these experiments demonstrate that propionylation could be monitored by IR spectroscopy by studying CH? stretching vibrations.
Chapter 6 highlights the effect of propionylation on the extent of cell death. In this chapter, it has been demonstrated that cell death caused with and without the use of cytotoxic agents involves propionylation. Thus, propionylation is basically a process involved in cell death and does not necessarily require an external cytotoxic agent to trigger cell death. However, at present, the significance of propionylation in cancer biology or pharmacology is not well known, but based on our findings and its importance in energy metabolism, it is expected that propionylation has a key role to play in the regulation of cell death.
Finally, Chapter 7 contains an overall summary of the thesis and discusses future directions of the research work carried out in this thesis. | |
