983 resultados para Molecular-scale periodicity


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Physical systems with co-existence and interplay of processes featuring distinct spatio-temporal scales are found in various research areas ranging from studies of brain activity to astrophysics. The complexity of such systems makes their theoretical and experimental analysis technically and conceptually challenging. Here, we discovered that while radiation of partially mode-locked fibre lasers is stochastic and intermittent on a short time scale, it exhibits non-trivial periodicity and long-scale correlations over slow evolution from one round-trip to another. A new technique for evolution mapping of intensity autocorrelation function has enabled us to reveal a variety of localized spatio-temporal structures and to experimentally study their symbiotic co-existence with stochastic radiation. Real-time characterization of dynamical spatio-temporal regimes of laser operation is set to bring new insights into rich underlying nonlinear physics of practical active- and passive-cavity photonic systems.

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The production of recombinant therapeutic proteins is an active area of research in drug development. These bio-therapeutic drugs target nearly 150 disease states and promise to bring better treatments to patients. However, if new bio-therapeutics are to be made more accessible and affordable, improvements in production performance and optimization of processes are necessary. A major challenge lies in controlling the effect of process conditions on production of intact functional proteins. To achieve this, improved tools are needed for bio-processing. For example, implementation of process modeling and high-throughput technologies can be used to achieve quality by design, leading to improvements in productivity. Commercially, the most sought after targets are secreted proteins due to the ease of handling in downstream procedures. This chapter outlines different approaches for production and optimization of secreted proteins in the host Pichia pastoris. © 2012 Springer Science+business Media, LLC.

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Over the past 50 years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modeling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics.

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Sol-gel-synthesized bioactive glasses may be formed via a hydrolysis condensation reaction, silica being introduced in the form of tetraethyl orthosilicate (TEOS), and calcium is typically added in the form of calcium nitrate. The synthesis reaction proceeds in an aqueous environment; the resultant gel is dried, before stabilization by heat treatment. These materials, being amorphous, are complex at the level of their atomic-scale structure, but their bulk properties may only be properly understood on the basis of that structural insight. Thus, a full understanding of their structure-property relationship may only be achieved through the application of a coherent suite of leading-edge experimental probes, coupled with the cogent use of advanced computer simulation methods. Using as an exemplar a calcia-silica sol-gel glass of the kind developed by Larry Hench, in the memory of whom this paper is dedicated, we illustrate the successful use of high-energy X-ray and neutron scattering (diffraction) methods, magic-angle spinning solid-state NMR, and molecular dynamics simulation as components to a powerful methodology for the study of amorphous materials.

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Phylogenetic analyses were performed on six genera and 46 species of the Neotropical palm tribe Geonomeae. The analyses were based on two low copy nuclear DNA sequences from the genes encoding phosphoribulokinase and RNA polymerase II. The basal node of the tribe was polytomous. Pholidostachys formed a monophyletic group. The currently accepted genera Calyptronoma and Calyptrogyne formed a well-supported clade with Calyptronoma resolved as paraphyletic to Calyptrogyne. Geonoma formed a strongly supported monophyletic group consisting of two main clades. ^ An evaluation of the genetic distinctness between Geonoma macrostachys varieties at a local and regional scale using inter-simple sequence repeat (ISSR) markers was performed. Clustering, ordination, and AMOVA suggested a lack of genetic distinctness between varieties at the regional level. A hierarchical AMOVA revealed that the genetic diversity mainly lies among the four localities sampled. A significant genetic differentiation between sympatric varieties occurred in one locality only. The current taxonomy of G. macrostachys, which recognizes only one species, was therefore supported. ^ The preferred habitat of sympatric G. macrostachys varieties with respect to edaphic, topographic, and light factors in three Peruvian lowland forests was studied. The two varieties were mostly encountered in different physiographically defined habitats, with variety acaulis occurring more often in floodplain forest and variety macrostachys in the tierra firme. Comparison of means tests revealed that nine to eleven of the 16 environmental variables were significantly different between varieties. Edaphic factors, mainly soil texture and K content, were better contributors than light conditions to distinguish the habitats occupied by the two varieties in all three study sites. It is concluded that habitat differentiation plays a role in the coexistence of these closely related species taxa. ^

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We measured the abundance of Cladium jamaicense (Crantz) seeds and three biomarkers in freshwater marsh soils in Shark River Slough (SRS), Everglades National Park (ENP) to determine the degree to which these paleoecological proxies reflect spatial and temporal variation in vegetation. We found that C. jamaicense seeds and the biomarkers Paq, total lignin phenols (TLP) and kaurenes analyzed from surface soils were all significantly correlated with extant aboveground C. jamaicense biomass quantified along a vegetation gradient from a C. jamaicense to a wet prairie/slough (WPS) community. Our results also suggest that these individual proxies may reflect vegetation over different spatial scales: Paq and kaurenes correlated most strongly (R 2 = 0.88 and 0.99, respectively) with vegetation within 1 m of a soil sample, while seeds and TLP reflected vegetation 0–20 m upstream of soil samples. These differences in the spatial scale depicted by the different proxies may be complementary in understanding aspects of historic landscape patterning. Soil profiles of short (25 cm) cores showed that downcore variation in C. jamaicense seeds was highly correlated with two of the three biomarkers (Paq, R 2 = 0.84, p<0.005; TLP, R 2 = 0.97, p<0.0001), and all four of the proxies indicated a recent increase in C. jamaicense biomass at the site. Using a preliminary depth-to-age relationship based on matching charcoal peaks with available ENP fire records (1980-present) specific to our coring site, we found that peak-depths in C. jamaicense seed concentration appeared to correspond to recent minimum water levels (e.g., 1989 and 2001), and low seed abundance corresponded to high water levels (e.g., 1995), consistent with the known autecology of C. jamaicense. In summary, the combination of C. jamaicense seeds and biomarkers may be useful for paleoecological reconstruction of vegetation change and ultimately in guaging the success of ongoing efforts to restore historic hydrologic conditions in the South Florida Everglades.

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CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

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Schistosomiasis is a neglected tropical disease that affects more than 200 million people worldwide. The main disease-causing agents, Schistosoma japonicum, S. mansoni and S. haematobium, are blood flukes that have complex life cycles involving a snail intermediate host. In Asia, S. japonicum causes hepatointestinal disease (schistosomiasis japonica) and is challenging to control due to a broad distribution of its snail hosts and range of animal reservoir hosts. In China, extensive efforts have been underway to control this parasite, but genetic variability in S. japonicum populations could represent an obstacle to eliminating schistosomiasis japonica. Although a draft genome sequence is available for S. japonicum, there has been no previous study of molecular variation in this parasite on a genome-wide scale. In this study, we conducted the first deep genomic exploration of seven S. japonicum populations from mainland China, constructed phylogenies using mitochondrial and nuclear genomic data sets, and established considerable variation between some of the populations in genes inferred to be linked to key cellular processes and/or pathogen-host interactions. Based on the findings from this study, we propose that verifying intraspecific conservation in vaccine or drug target candidates is an important first step toward developing effective vaccines and chemotherapies against schistosomiasis.

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In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.

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The fruit is one of the most complex and important structures produced by flowering plants, and understanding the development and maturation process of fruits in different angiosperm species with diverse fruit structures is of immense interest. In the work presented here, molecular genetics and genomic analysis are used to explore the processes that form the fruit in two species: The model organism Arabidopsis and the diploid strawberry Fragaria vesca. One important basic question concerns the molecular genetic basis of fruit patterning. A long-standing model of Arabidopsis fruit (the gynoecium) patterning holds that auxin produced at the apex diffuses downward, forming a gradient that provides apical-basal positional information to specify different tissue types along the gynoecium’s length. The proposed gradient, however, has never been observed and the model appears inconsistent with a number of observations. I present a new, alternative model, wherein auxin acts to establish the adaxial-abaxial domains of the carpel primordia, which then ensures proper development of the final gynoecium. A second project utilizes genomics to identify genes that regulate fruit color by analyzing the genome sequences of Fragaria vesca, a species of wild strawberry. Shared and distinct SNPs among three F. vesca accessions were identified, providing a foundation for locating candidate mutations underlying phenotypic variations among different F. vesca accessions. Through systematic analysis of relevant SNP variants, a candidate SNP in FveMYB10 was identified that may underlie the fruit color in the yellow-fruited accessions, which was subsequently confirmed by functional assays. Our lab has previously generated extensive RNA-sequencing data that depict genome-scale gene expression profiles in F. vesca fruit and flower tissues at different developmental stages. To enhance the accessibility of this dataset, the web-based eFP software was adapted for this dataset, allowing visualization of gene expression in any tissues by user-initiated queries. Together, this thesis work proposes a well-supported new model of fruit patterning in Arabidopsis and provides further resources for F. vesca, including genome-wide variant lists and the ability to visualize gene expression. This work will facilitate future work linking traits of economic importance to specific genes and gaining novel insights into fruit patterning and development.

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The thermoset epoxy resin EPON 862, coupled with the DETDA hardening agent, are utilized as the polymer matrix component in many graphite (carbon fiber) composites. Because it is difficult to experimentally characterize the interfacial region, computational molecular modeling is a necessary tool for understanding the influence of the interfacial molecular structure on bulk-level material properties. The purpose of this research is to investigate the many possible variables that may influence the interfacial structure and the effect they will have on the mechanical behavior of the bulk level composite. Molecular models are established for EPON 862-DETDA polymer in the presence of a graphite surface. Material characteristics such as polymer mass-density, residual stresses, and molecular potential energy are investigated near the polymer/fiber interface. Because the exact degree of crosslinking in these thermoset systems is not known, many different crosslink densities (degrees of curing) are investigated. It is determined that a region exists near the carbon fiber surface in which the polymer mass density is different than that of the bulk mass density. These surface effects extend ~10 Å into the polymer from the center of the outermost graphite layer. Early simulations predict polymer residual stress levels to be higher near the graphite surface. It is also seen that the molecular potential energy in polymer atoms decreases with increasing crosslink density. New models are then established in order to investigate the interface between EPON 862-DETDA polymer and graphene nanoplatelets (GNPs) of various atomic thicknesses. Mechanical properties are extracted from the models using Molecular Dynamics techniques. These properties are then implemented into micromechanics software that utilizes the generalized method of cells to create representations of macro-scale composites. Micromechanics models are created representing GNP doped epoxy with varying number of graphene layers and interfacial polymer crosslink densities. The initial micromechanics results for the GNP doped epoxy are then taken to represent the matrix component and are re-run through the micromechanics software with the addition of a carbon fiber to simulate a GNP doped epoxy/carbon fiber composite. Micromechanics results agree well with experimental data, and indicate GNPs of 1 to 2 atomic layers to be highly favorable. The effect of oxygen bonded to the surface of the GNPs is lastly investigated. Molecular Models are created for systems with varying graphene atomic thickness, along with different amounts of oxygen species attached to them. Models are created for graphene containing hydroxyl groups only, epoxide groups only, and a combination of epoxide and hydroxyl groups. Results show models of oxidized graphene to decrease in both tensile and shear modulus. Attaching only epoxide groups gives the best results for mechanical properties, though pristine graphene is still favored.

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Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

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In this work we compare Grapholita molesta Busck (Lepidoptera: Tortricidae) populations originated from Brazil, Chile, Spain, Italy and Greece using power spectral density and phylogenetic analysis to detect any similarities between the population macro- and the molecular micro-level. Log-transformed population data were normalized and AR(p) models were developed to generate for each case population time series of equal lengths. The time-frequency/scale properties of the population data were further analyzed using wavelet analysis to detect any population dynamics frequency changes and cluster the populations. Based on the power spectral of each population time series and the hierarchical clustering schemes, populations originated from Southern America (Brazil and Chile) exhibit similar rhythmic properties and are both closer related with populations originated from Greece. Populations from Spain and especially Italy, have higher distance by terms of periodic changes on their population dynamics. Moreover, the members within the same cluster share similar spectral information, therefore they are supposed to participate in the same temporally regulated population process. On the contrary, the phylogenetic approach revealed a less structured pattern that bears indications of panmixia, as the two clusters contain individuals from both Europe and South America. This preliminary outcome will be further assessed by incorporating more individuals and likely employed a second molecular marker.

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Marine mussels are exceptionally well-adapted to live in transitional habitats where they are exposed to fluctuating environmental parameters and elevated levels of natural and anthropogenic stressors throughout their lifecycle. However, there is a dearth of information about the molecular mechanisms that assist in dealing with environmental changes. This project aims to investigate the molecular mechanisms governing acclimatory and stress responses of the Mediterranean mussel (Mytilus galloprovincialis) by addressing relevant life stages and environmental stressors of emerging concern. The experimental approach consisted of two phases to explore (i) the physiological processes at early life history and the consequences of plastic pollution and (ii) the adult physiology processes under natural habitats. As the first phase, I employed a plastic leachate (styrene monomer), and polystyrene microplastics to understand the modulation of cytoprotective mechanisms during the early embryo stages. Results revealed the onset of transcriptional impairments of genes involved in MXR-related transporters and other physiological processes induced by styrene and PS-MPs. In the second phase, as a preliminary analysis, microbiota profile of adult mussels at the tissue scale and its surrounding water was explored to understand microbiota structures that may reflect peculiar adaptations to the respective tissue functions. The broader experiment has been implemented to understand the variability of transcriptional profiles in the mussel digestive glands in the natural setting. All the genes employed in this study have shown possibilities to use as molecular biomarker responses throughout the year for monitoring the physiology of mussels living in a particular environment and, in turn, more properly detecting changes in the environment. As a whole, my studies provide insights into the interactions between environmental parameters, and intrinsic characters, and physiology of marine bivalves, and it could help to interpretation of responses correctly under stress conditions and climate change scenarios.

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The study of the spectroscopic phenomena in organic solids, in combination with other techniques, is an effective tool for the understanding of the structural properties of materials based on these compounds. This Ph.D. work was dedicated to the spectroscopic investigation of some relevant processes occurring in organic molecular crystals, with the goal of expanding the knowledge on the relationship between structure, dynamics and photoreactivity of these systems. Vibrational spectroscopy has been the technique of choice, always in combination with X-ray diffraction structural studies and often the support of computational methods. The vibrational study of the molecular solid state reaches its full potential when it includes the low-wavenumber region of the lattice-phonon modes, which probe the weak intermolecular interactions and are the fingerprints of the lattice itself. Microscopy is an invaluable addition in the investigation of processes that take place in the micro-meter scale of the crystal micro-domains. In chemical and phase transitions, as well as in polymorph screening and identification, the combination of Raman microscopy and lattice-phonon detection has provided useful information. Research on the fascinating class of single-crystal-to-single-crystal photoreactions, has shown how the homogeneous mechanism of these transformations can be identified by lattice-phonon microscopy, in agreement with the continuous evolution of their XRD patterns. On describing the behavior of the photodimerization mechanism of vitamin K3, the focus was instead on the influence of its polymorphism in governing the product isomerism. Polymorphism is the additional degree of freedom of molecular functional materials, and by advancing in its control and properties, functionalities can be promoted for useful applications. Its investigation focused on thin-film phases, widely employed in organic electronics. The ambiguities in phase identification often emerging by other experimental methods were successfully solved by vibrational measurements.