Identifying human diamine sensors for death related putrescine and cadaverine molecules

Pungent chemical compounds originating from decaying tissue are strong drivers of animal behavior. Two of the best-characterized death smell components are putrescine (PUT) and cadaverine (CAD), foul-smelling molecules produced by decarboxylation of amino acids during decomposition. These volatile polyamines act as 'necromones', triggering avoidance or attractive responses, which are fundamental for the survival of a wide range of species. The few studies that have attempted to identify the cognate receptors for these molecules have suggested the involvement of the seven-helix trace amine-associated receptors (TAARs), localized in the olfactory epithelium. However, very little is known about the precise chemosensory receptors that sense these compounds in the majority of organisms and the molecular basis of their interactions. In this work, we have used computational strategies to characterize the binding between PUT and CAD with the TAAR6 and TAAR8 human receptors. Sequence analysis, homology modeling, docking and molecular dynamics studies suggest a tandem of negatively charged aspartates in the binding pocket of these receptors which are likely to be involved in the recognition of these small biogenic diamines

Are polyamines detected by the TAAR receptor family in the human Choroid Plexus?

Choroid plexuses (CPs) are highly vascularized structures located at the ventricular system of the brain constituted by a monolayer of choroid plexus epithelial cells (CPEC) located on a basement membrane which form a barrier between the blood and the cerebrospinal fluid (CSF) (BCSFB). CPECs through their interconnections restrict the passage of different substances and pathogens between the blood and the CSF. Moreover, the microvilli at the apical side and an extensive infolding at the basolateral side enhance the contact between the CP epithelium and CSF, and between the epithelium and stroma interstitial fluid, respectively, thus allowing one of its main functions which is to maintain cerebral homeostasis. Among other features, there is the chemical vigilance which is performed by different mechanisms as the taste signalling, the clearance of xenobiotics, the clearance of amyloid beta and finally but with more relevance to this work, the olfactory signalling. This olfactory cascade is activated when an odorant binds to an olfactory receptor (OR) which is a G protein-coupled receptor (GPCR) that exists in different tissues in mammalian´s body. The trace amine-associated receptors (TAARs) are a family of rhodopsin-like GPCRs which act as olfactory receptors with TAAR1 exception and are expressed in different tissues of different species. In humans there are 6 different types of TAARs and 3 pseudogenes. TAAR 13c, identified in zebrafish, can bind to cadaverine, a polyamine. Polyamines are polycations which appear naturally in cells and are closely related with neuronal cell biochemical activity at different points. Cadaverine is a diamine compound produced by the putrefaction of animal tissue and enable an aversive response to this odour. A previous data base research showed that TAAR1, 2 and 5 are the human receptors with more homology with TAAR13c and one of the more abundant TAARs in humans, which probably can trigger a response to a polyamine stimulus. TAAR1 was selected for this study because we could not amplify TAAR2 and 5 by RT-PCR in HIBCPP cell line. The HIBCPP cell line from choroid plexus carcinoma was used to the experiments and the expression of the mRNAs to the TAAR1 was demonstrated by the RT-PCR technique. In respect to polyamine´s biosynthesis and catabolic pathway enzymes identification, the RT-PCR technique enabled the confirmation of their mRNA expression after Sanger sequencing. The expression of key enzymes to these pathways was also demonstrated by immunocytochemistry and Western blot, in human CP cell line. To test the cell´s response to polyamines, calcium imaging (Ca2+ imaging) assays were performed and showed clear responses to cadaverine. After these tests, silencing of TAAR1 with a specific small interfering RNA (siRNA) was done and preliminary Ca2+ imaging responses to cadaverine enable to see a decrease in CP cells response, highlighting the potential role of TAAR1 in this polyamine response. In the closer future, more silencing assays will be done and Ca2+ imaging with transfected cells will be done too with cadaverine and other polyamines already tested as spermine, spermidine and putrescine. Western blot and immunocytochemistry techniques will enable the TAAR1 protein characterization and localization in the HIBCPP cells.No sistema nervoso central (SNC) é possível observar a presença de distintas barreiras que assumem um papel fundamental na proteção do cérebro contra as variações nas concentrações de determinadas substâncias ao nível do sangue, assim como contra a entrada de organismos patogénicos, toxinas e outras substâncias. Contudo, este nem sempre é um mecanismo de controlo favorável uma vez que estas podem funcionar como uma barreira à entrada de substâncias para o tratamento de patologias do SNC. De entre as barreiras existentes no cérebro salienta-se a barreira entre o sangue e o cérebro, a barreira aracnóideia e a barreira entre o sangue e o líquido cefalorraquidiano, o plexo coroide. O plexo coroide (CP) é uma estrutura altamente vascularizada localizada em cada um dos quatro ventrículos cerebrais. O CP é constituído por uma monocamada de células epiteliais localizada numa membrana basal e que por sua vez, formam uma barreira entre o sangue e o líquido cefalorraquidiano. Estas células, através das suas interconexões como é o caso das junções aderentes, junções apertadas e desmossomas, impedem a passagem de diferentes substâncias e organismos patogénicos, do sangue para o líquido cefalorraquidiano. Para além destas evidências, há a destacar o facto de as microvilosidades presentes na face apical assim como as invaginações existentes na superfície basal permitirem também uma boa interação entre o epitélio do plexo e o líquido cefalorraquidiano e entre o epitélio e o fluido intersticial, respetivamente, permitindo um conjugar de condições que vão assegurar uma correta manutenção da homeostase cerebral. De entre outras funções desempenhadas por esta estrutura, salienta-se a produção e secreção do líquido cefalorraquidiano e a vigilância química deste, que é assegurada por diferentes mecanismos como a sinalização gustativa, a remoção de xenobióticos, a remoção da proteína ß-amilóide, envolvida em doenças como a doença de Alzheimer, e por fim a sinalização olfativa. Esta cascata de sinalização olfativa é ativada quando uma molécula odorante se liga a um recetor olfativo, um recetor acoplado a uma proteína G. Esta ligação vai despoletar as cascatas de segundo mensageiro da adenosina monofosfato cíclica e do inositol 1,4,5-trifosfato. Há evidências de que a estimulação destes recetores olfativos ocorre pela ativação da proteína G olfativa levando a um aumento da adenosina monofosfato cíclica através da adenilato ciclase 3 de membrana, resultando num aumento do influxo de Ca2+ através de um canal nucleotídico cíclico, resultando na despolarização da membrana e consequentemente na geração de um potencial de ação nos neurónios olfativos. Os trace amine-associated receptors (TAARs) são uma família de recetores acoplados a proteína G que, à exceção do TAAR1, atuam como recetores olfativos, sendo expressos em diferentes tecidos em diferentes espécies. Diferentes estudos permitiram comprovar a expressão destes recetores em tecidos do epitélio olfativo e noutros como é o caso do cérebro, medula espinhal, coração, pulmões, fígado, baço, trato gastrointestinal, rins, testículos e células sanguíneas como os leucócitos. Em humanos é possível identificar 6 tipos diferentes de TAARs (1,2,5,6,8 e 9) e 3 pseudogenes (3, 4 e 7). Os genes que codificam para estes recetores encontram-se localizados no cromossoma 6 q23.1. O TAAR13c, expresso no peixe-zebra foi identificado como sendo o responsável por ligar à cadaverina, uma poliamina resultante da decomposição de tecidos mortos, gerada através da descarboxilação da lisina pela enzima lisina-descarboxílase, que ao ser detetada por este peixe, despoleta uma resposta aversiva por parte deste. As poliaminas são policatiões presentes em todas as células, que interagem com moléculas carregadas negativamente como é o caso do ácido desoxirribonucleico (DNA), ácido ribonucleico (RNA) ou ainda as proteínas. Estas substâncias como é o caso da cadaverina, espermina, espermidina e putrescina participam em diversas funções a nível fisiológico como o crescimento celular, proliferação, diferenciação, transcrição, tradução, regulação de canais iónicos, atividade enzimática, constituição das interações célula-célula e resposta ao stress oxidativo. Estas duas últimas funções conferem às poliaminas um papel fundamental no surgimento de diversas patologias do foro neurológico como é o caso da doença Parkinson, da esclerose lateral amiotrófica e isquemia, em que os valores destas substâncias surgem alterados. Contudo as poliaminas ao controlarem o stress oxidativo vão também ter um papel benéfico no controlo da doença de Alzheimer. Com o objetivo de estabelecer homologia entre o TAAR13c do peixe-zebra e os TAARs humanos foi realizada uma pesquisa com a ferramenta BlastX (NCBI) que revelou a existência de diferentes TAAR com maior homologia com o TAAR13c. Após uma pesquisa na base de dados Gene Expression Omnibus, foram selecionados para o estudo, os TAAR1, 2 e 5 por estes apresentarem maior abundância em humanos. Estes dados levaram-nos a supor que estes recetores teriam capacidade para despoletar uma resposta em células humanas do plexo coroide quando estimuladas por poliaminas como a cadaverina. Assim, o uso de uma linha de células humanas de carcinoma do plexo coroide, através da técnica de RT-PCR seguida de sequenciação pelo método de Sanger, permitiu identificar a expressão do mRNA para o TAAR1, o que não se verificou para os TAAR2 e 5. Um processo idêntico foi utilizado para a análise da expressão dos mRNAs para as enzimas das vias anabólica e catabólica das poliaminas, sendo a sua expressão confirmada por sequenciação. As enzimas chave destas vias, a espermina sintetase e a ornitina descarboxílase, foram também analisadas através das técnicas de Western blot e imunocitoquímica. Após a realização de um ensaio de MTT que permitiu comprovar que os estímulos de cadaverina aplicados não iriam comprometer a viabilidade celular, foram realizados ensaios de calcium imaging que possibilitaram verificar respostas por parte das células após estimulação com diferentes concentrações de cadaverina. Por forma e testar a especificidade da ligação do TAAR1 à cadaverina, foi realizado um ensaio preliminar (n=1) de silenciamento, transfetando as células com um pequeno RNA de interferência (siRNA) para o TAAR1. Os resultados demonstraram uma diminuição das respostas celulares à cadaverina quando em comparação com os controlos feitos apenas com o agente de transfeção ou com este último em conjunto com um scramble siRNA. Estes resultados, embora preliminares, são promissores quanto ao papel do TAAR1 na deteção da cadaverina. Futuramente, serão realizados novos ensaios de silenciamento do TAAR1 e estímulos com outras poliaminas como a espermina, espermidina e putrescina. Serão também efetuados ensaios de Western blot e imunocitoquímica, permitindo avaliar a expressão e localização do TAAR1

The Bouquet of Death and Decay

This text talks about death and the smell of death. It elaborates on the difficulties that arise in attempting to describe smells and olfactory phenomena in a visual essay using images and words. The ineffable qualities of smell and death prevent certainties. Materiality, however, can provide a window into awareness and the beginnings of understanding

Olfactory coding in vertebrates: a novel tuning mechanism for receptor affinity and evolution of the olfactory receptor repertoire

Information about our environment is to a large extent carried by the chemical senses, and in particular the olfactory sense. Vertebrates perceive thousands of diverse odor molecules with a supply of a wide range of essential information ranging from localising prey or food, avoiding predators, mating behaviour, to social communication. Because olfactory receptor proteins play such an essential role in the specific recognition of diverse stimuli, understanding how they interact with and transduce their cognate ligands is a high priority. This constitutes one of the most complex ligand/receptor binding problems in biology due to the sheer quantity of potential odor molecules facing a limited albeit huge number of different olfactory receptors. Most olfactory receptors are G-protein coupled receptors and form large gene families. One type of olfactory receptors is the trace amine-associated receptor family (TAAR). TAARs generally recognize amines and one particular member of the zebrafish TAAR family, TAAR13c, is a high affinity receptor for the death-associated odor cadaverine, which induces aversive behavior. Here we have modeled the cadaverine/TAAR13c interaction by multistep docking. By exchanging predicted binding residues via site-directed mutagenesis, and measuring the activity of the mutant receptors, we confirmed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. Elimination of the external binding site generated supersensitive receptors which suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. Potentially related mechanisms have been described for non-olfactory G-protein coupled receptors. The topology of TAAR-expressing neurons in the teleost olfactory epithelium has not been described yet. We have investigated representative taar genes from three classes to test the principle of partial spatial segregation known from other olfactory receptor families for the TAAR family. We report that expression of taar genes is intermingled with expression zones of odorant receptor genes, which in fish share a single sensory surface with TAARs. Individual taar genes show distinct, albeit broadly overlapping expression zones. In the third part of my thesis I investigated the genome of a cartilaginous fish, Scyliorhinus canicula, commonly known as small spotted catshark in order to delineate its chemosensory receptor repertoire: OR, V1R/V2R, TAAR, and T1R/T2R. This is the first repertoire described for a true shark, an important intermediate in the evolution of vertebrates. In contrast to bony vertebrates, but very similar to a chimera (elephant shark), the olfactory receptor repertoire of catshark is dominated by the V2R family

The Development of a MWCNT-DAO Biosensor for the Detection of Cadaverine in the Assessment of Periodontal Disease

Periodontal disease is one the most common afflictions of human populations and is a major challenge in both the developed and developing world. Cadaverine is a biomolecule which has implications in a myriad of human diseases, in particular periodontal disease. Porphyromonas gingivalis has been suggested as one of the key microorganisms in periodontal disease, and its pathogenesis has been extensively researched. It has a number of metabolites which contribute towards its pathogenicity, and among them, cadaverine has seen particularly detrimental effects in both oral and systemic health. Traditional testing methods for periodontal disease do not provide clinicians with active disease state measurements, but instead provide information on the history of the disease. Thus, the development of a real-time biosensor, providing point-of-care information regarding the metabolite status of the oral cavity would be of significant impact for oral disease diagnostics. This thesis reports the development and testing of a newly designed working electrode fitted to a flexible screen-printed sensor platform for the detection of the biogenic amine, cadaverine. The results in chapter 3 showed that DAO enhanced MWCNT dispersion and increased overall electrode surface topographies, resulting in a more wettable electrode for faster, more efficient electron transfer kinetics. Scanning electron microscopy, UV-Vis, Fourier transform intra-red spectroscopy, raman spectroscopy, and energy diffraction x-ray spectroscopy confirmed the functional group changes the MWCNT’s underwent during modification. Investigations into the modified biosensors heterogenous electron transfer rates were carried out and determined an increase to the peak to peak separation, possible due to the additional modification layers on the electrode surface, and the presence of a binder. Chapter 4 investigated electrochemical efficacy of the MWCNT-DAO biosensor against cadaverine in stock solutions, and artificially simulated saliva. The biosensor demonstrated positive concentration dependant correlations towards cadaverine from a range of 3 µg/ml to 150 µg/ml. Similarly in artificial saliva, the biosensors efficacy remained consistent, and presented a potential for the biosensor to function outside of stock sample solutions. The cytotoxic effects of P. gingivalis and cadaverine were investigated in Chapter 5. Initial monocyte differentiation into macrophages was confirmed using flow cytometry. Scanning electron microscopy was used to demonstrate the phagocytic effects of macrophages towards P. gingivalis and resulted in phagocytosis being visualised by the pseudopodia-like appendages engulfing the bacterium. Cell viability, and cell migration assays, and showed significant reductions in the viability and migratory effects of human epithelial keratinocyte and M0 macrophage cells in response to a P. gingivalis infection, and as a response to increasing cadaverine levels, above the physiological normal thresholds. The findings from this study present a MWCNT-DAO biosensor which was able to detect cadaverine at concentrations which are respective to those in periodontal disease, in stock solutions and in simulated human saliva. Furthermore, the cytotoxic effects of cadaverine and its precursor bacteria, P. gingivalis were elucidated, and showed significant detrimental effects towards human macrophage and human epithelial keratinocyte cells. thus, the biosensor developed in this study may be used as a tool for determining the extent of a patient’s periodontal disease, using a rapid, cost effective and point-of-care biosensor

Direct Detection of Biogenic Amines from Fish

The biogenic amines content in fish products has been widely studied due to their potential toxicity and possible indication of the spoilage degree of food. One particular amine, histamine, is behind several outbreaks of food poisoning, particularly in fish products. However, histamine alone appears to be insufficient to cause intoxications, being putrescine and cadaverine, potentiators agents that contribute to its toxicity. Several methodologies have been studied and developed for determination of bi-ogenic amines in food products. Ion Mobility Spectrometry coupled with Multi Capil-lary Columns or Gas Chromatography offers a higher sensitivity and selectivity on com-plex biological matrices analysis, providing the monitoring of trace levels of volatile compounds. The aim of this thesis is to evaluate Gas Chromatography coupled with Ion Mobil-ity Spectrometry as a tool for monitoring non-volatile amines emission from fish tissues matrices, allowing the detection and establishment of specific patterns of biogenic amines. Samples of histamine dihydrochloride, putrescine, cadaverine, tyramine, trypta-mine, spermine, spermidine and phenethylamine were analysed. Samples of atlantic bo-nito, atlantic horse mackerel and sardine were collected and analysed over time of four days to allow an assessment of fish spoilage. With the exception of histamine, it was possible to obtain an identifier pattern for all analysed amines. The presence of some amines was also observed in the fish samples spectra

The teleost taar family of olfactory receptors: From rapidly evolving receptor genes to ligand-induced behavior

Trace amine-associated receptors (TAARs) have recently been shown to function as olfactory receptors in mammals. In this current study, the taar gene family has been delineated in jawless, cartilaginous, and bony fish (zero, 2, and >100 genes, respectively). I conclude that the taar genes are evolutionary much younger than the related OR and ORA/V1R olfactory receptor families, which are present already in lamprey, a jawless vertebrate. The 2 cartilaginous fish genes appear to be ancestral for 2 taar classes, each with mammalian and bony fish (teleost) representatives. Unexpectedly, a whole new clade, class III, of taar genes originated even later, within the teleost lineage. Taar genes from all 3 classes are expressed in subsets of zebrafish olfactory receptor neurons, supporting their function as olfactory receptors. The highly conserved TAAR1 (shark,mammalian, and teleost orthologs) is not expressed in the olfactory epithelium and may constitute the sole remnant of a primordial, non olfactory function of this family. Class III comprises three-fourths of all teleost taar genes and is characterized by the complete loss of the aminergic ligand-binding motif, stringently conserved in all 25 genes of the other 2 classes. Two independent intron gains in class III taar genes represent extraordinary evolutionary dynamics, considering the virtual absence of intron gains during vertebrate evolution. The dN/dS analysis suggests both minimal global negative selection and an unparalleled degree of local positive selection as another hallmark of class III genes. The accelerated evolution of class III teleost taar genes conceivably might mark the birth of another olfactory receptor gene family. Ligands have only been identified for a handful of olfactory receptors of mammals and insects, while only a single teleost olfactory receptor have been deorphanized, a member of the OlfC family, OlfCa. Zebrafish TAAR olfactory receptors of classI are good candidates for having amines as possible ligands, due to the presence of the aminergic ligand binding motifs. This study identifies diamines as specific ligands for a taar receptor, DrTAAR13c. These diamines activate a sparse subset of olfactory sensory neurons, as indicated by c-Fos expression in olfactory epithelium. Diamines, putrescine and cadaverine, are foul-smelling aliphatic polycations that occur naturally as a result of bacterial decarboxylation of amino acids lysine and arginine, respectively. The 15 concentration of diamines in their environment is correlated to the degree of putrefication. In the behavioral assay, zebrafish exposed to even low concentration of diamines show dramatic, quantifiable aversion, while it shows attraction towards food stimulus and no response for water. The ligand spectrum of TAAR13c closely parallels the behavioral effectiveness of these diamines. This data is consistent with the existence of a defined neuronal microcircuit that elicits a characteristic behavior upon activation of a single olfactory receptor, a novum in the vertebrate sense of smell

FRSH: A Frugal and Rapid bioSensor for in-House use to assess meat spoilage

Biogenic amines (BAs) represent a toxicological risk in many food products. Putrescine is the most common BAs found in food and is frequently used as a marker for food quality. Today there is a lack of regulation concerning safe putrescine limits in food as well as outdated food handling practices leading to unnecessary putrescine intake. Conventional methods used to evaluate BAs in food are generally time and resource heavy, limiting the options for on-site analysis. In this work, we developed a transcription-factor based biosensor for quantification of putrescine, one of the BAs in meat products, using a naturally occurring putrescine responsive repressor-operator pair (PuuR-puuO) from Escherichia coli. Moreover, we demonstrate the use of the putrescine biosensor with a paper-based cell-free device that enables low-cost and rapid putrescine detection. The system was validated using a variety of consumer meat samples with comparable performance to standard well-plate analysis methods. We propose that our system is ready for use to assess the safety of meat products will contribute to a new phase of low-cost biosensors designed for food safety

Biogenic Amines as a Product of the Metabolism of Proteins in Beer

For many years the only beer that was commercially available in the United States were simple lagers and ales made primarily of barley (and other cereals), water, hops, and yeast. These beer varieties were simple and quick to produce with high levels of consistency. In the last 30 years the rise in craft and microbreweries have dramatically changed the landscape of brewing and beer. As smaller breweries rose in popularity so did the use of unique ingredients such as vegetables, fruits, herbs, meat, etc. along with higher gravity mashes and long barrel aging times. All these ingredients have a profound effect on the chemical makeup of a beer, adding a variety of organic acids, antimicrobials, amino acids, etc. These compounds become important when examining the influences of what may be the most important ingredient in beer (and all alcoholic beverages), yeast. Yeast, specifically Saccharomyces yeast (though many other genera are capable of this), consume simple carbohydrates within the unfermented beer and produce carbon dioxide and alcohol. While these fermentation biproducts are the most well-known compounds produced by yeast, there are many others. During development (log phase) and times of stress/carbohydrate starvation, yeast can often consume proteins and amino acids and produce biogenic amines. Biogenic amines (BA’s) are small nitrogenous bases produced through the enzymatic decarboxylation of amino acids by living organisms. Examples of biogenic amines include histamine, serotonin, tyramine, and spermine (though many others exist). These compounds are important and powerful signaling molecules used by every living creature on Earth. While endogenous biogenic amines produced within the body are critical to survival, exogenous biogenic amines in a consumed food product can be highly dangerous; biogenic amines can cause a variety of symptoms ranging from headaches to hyper/hypotension to pseudo-anaphylaxis. While all living things create these compounds, the levels in most food products are too low to cause any symptoms: The only products with potentially dangerous levels of biogenic amines are fermented foods as bacteria and fungi used to make these products can also produce biogenic amines. While the biogenic amine content in certain fermented foods like cheese, sausages, and lacto-fermented vegetables has been well studied, formation of biogenic amines in beer remains largely under-examined. The three main objectives of this research were: 1., to perform an extensive review of the literature to ascertain analytical methods, types, levels, and sources of biogenic amines in beer, 2., to develop a quick and effective high performance liquid chromatography (HPLC) method to examine the biogenic amine content in beer, and 3., use this HPLC method to test a variety of commercial beer sample to examine what effect (if any) these new microbrewery recipes had on the biogenic amine profile of the final beverage. Method development focused primarily on the tagging of BA’s using the Waters AccQ Rapid Amine Tag (AccQ Tag Ultra, 2014) and analysis using an Agilent Organic Acids column. In total, more than 17 HPLC methodologies were tested and only one effectively resolved a mixed amine standard solution. Despite this successful test using a mixed standard, no mobile phase adjustment was able to effectively separate biogenic amines from interfering compounds (free amino acids or other nitrogenous compounds) in a beer sample. It was ultimately decided that moving forward, an effective extraction method, involving sample cleanup and capable of separating biogenic amines from highly soluble peptides and amino acids would be necessary for successful analysis

Study of New Antibacterial Packaging Systems through the Monitoring of Shelf Life Markers.

The specific objectives of this project were: 1) to characterize the preservation efficiency - biogenic amines and microbial load- of commercial available packaging materials (multilayer packaging with metal layer) in 8 different ways on raw and cooked ham; 2) to study the correlation between biogenic amines and microorganisms, ATP and microorganisms in different foods; to select the appropriate shelf life markers of raw chicken meat and ham; 3) to select probiotic bacteria with potential food application and optimize fermentation conditions; 4) to obtain the effectiveness of ATP as a shelf life marker in modified packaging, and to examine L. plantarum cell-free supernatant to be used as part of antibacterial packaging for extending ham shelf-life; 5) to prepare and evaluate different prototypes PLA/paper packages with addition of graphene-based composite, probiotic bacteria – L. plantarum IMC 509 - on raw chicken meat. The general introduction (Chapter 1) provides a general background in concept of antibacterial packaging and shelf life, the important role of packaging and biological indicators. The feasibility of Lactic acid bacteria and graphene as bacteriostatic agents for improved packaging were highlighted, as well as the application background of BA and ATP as shelf life markers. Chapter 2 – formation of biogenic amines and growth of spoilage-related microorganisms in the refrigerated ham with different commercial packages; Chapter 3 – determination of ATP-related compounds in dry-cured ham by HPLC; Chapter 4 – study on the antibacterial activity and fermentation of Lactic acid bacteria and metabolites; Chapter 5 – determination of ATP-related compounds by HPLC to study the effect of cell-free supernatants of Lactiplantibacillus plantarum on the shelf life of sliced dry-cured ham; Chapter 6 – assessment of shelf-life of chicken breast meat stored in the novel composite graphene and probiotic paper-PLA packaging; Chapter 7 – general discussion and summary