Generalized atoms in molecules approach (GAIM approach)

This thesis involves two parts. The first is a new-proposed theoretical approach called generalized atoms in molecules (GAIM). The second is a computational study on the deamination reaction of adenine with OH⁻/nH₂O (n=0, 1, 2, 3) and 3H₂O. The GAIM approach aims to solve the energy of each atom variationally in the first step and then to build the energy of a molecule from each atom. Thus the energy of a diatomic molecule (A-B) is formulated as a sum of its atomic energies, EA and EB. Each of these atomic energies is expressed as, EA = Hᴬ + Vₑₑᴬᴬ + 1/2Vₑₑᴬ<>ᴮ EB = Hᴮ + Vₑₑᴮᴮ + 1/2Vₑₑᴬ<>ᴮ where; Hᴬ and Hᴮ are the kinetic and nuclear attraction energy of electrons of atoms A and B, respectively; Vₑₑᴬᴬ and Vₑₑᴮᴮ are the interaction energy between the electrons on atoms A and B, respectively; and Vₑₑᴬ<>ᴮ is the interaction energy between the electrons of atom A with the electrons of atom B. The energy of the molecule is then minimized subject to the following constraint, |ρA(r)dr + |ρB(r)dr = N where ρA(r) and ρB(r) are the electron densities of atoms A and B, respectively, and N is the number of electrons. The initial testing of the performance of GAIM was done through calculating dissociation curves for H₂, LiH, Li₂, BH, HF, HCl, N₂, F₂, and Cl₂. The numerical results show that GAIM performs very well with H₂, LiH, Li₂, BH, HF, and HCl. GAIM shows convergence problems with N₂, F₂, and Cl₂ due to difficulties in reordering the degenerate atomic orbitals Pₓ, Py, and Pz in N, F, and Cl atoms. Further work for the development of GAIM is required. Deamination of adenine results in one of several forms of premutagenic lesions occurring in DNA. In this thesis, mechanisms for the deamination reaction of adenine with OH⁻/nH₂O, (n = 0, 1, 2, 3) and 3H₂O were investigated. HF/6-31G(d), B3LYP/6-31G(d), MP2/6-31G(d), and B3LYP/6-31+G(d) levels of theory were employed to optimize all the geometries. Energies were calculated at the G3MP2B3 and CBS-QB3 levels of theory. The effect of solvent (water) was computed using the polarizable continuum model (PCM). Intrinsic reaction coordinate (IRC) calculations were performed for all transition states. Five pathways were investigated for the deamination reaction of adenine with OH⁻/nH₂O and 3H₂O. The first four pathways (A-D) begin with by deprotonation at the amino group of adenine by OH⁻, while pathway E is initiated by tautomerization of adenine. For all pathways, the next two steps involve the formation of a tetrahedral intermediate followed by dissociation to yield products via a 1,3-hydrogen shift. Deamination with a single OH⁻ has a high activation barrier (190 kJ mol⁻¹ using G3MP2B3 level) for the rate-determining step. Addition of one water molecule reduces this barrier by 68 kJ mol⁻¹ calculated at G3MP2B3 level. Adding more water molecules decreases the overall activation energy of the reaction, but the effect becomes smaller with each additional water molecule. The most plausible mechanism is pathway E, the deamination reaction of adenine with 3H₂O, which has an overall G3MP2B3 activation energy of 139 and 137 kJ mol⁻¹ in the gas phase and PCM, respectively. This barrier is lower than that for the deamination with OH⁻/3H₂O by 6 and 2 kJ mol⁻¹ in the gas phase and PCM, respectively.

Design and Synthesis of Acridine-Based Macrocyclic G-Quadruplex DNA Ligands

DNA sequences that are rich in the guanine nucleic base possess the ability to fold into higher order structures called G-quadruplexes. These higher level structures are formed as a result of two sets of four guanine bases hydrogen-bonding together in a planar arrangement called a guanine quartet. Guanine quartets subsequently stack upon each other to form quadruplexes. G-quadruplexes are mainly localized in telomeres as well as in oncogene promoters. One unique and promising therapeutic approach against cancer involves targeting and stabilizing G-quadruplexes with small molecules, generally in order to suppress oncogene expression and telomerase enzyme activity; the latter has been found to contribute to “out-of control” cell growth in ca. 80-85% of all cancer cells and primary tumours while being absent in normal somatic cells. In this work, we present efforts towards designing and synthesizing acridine-based macrocycles (Mh) and (Mb) with the purpose of providing potential G4 ligands that are suited for selective binding to G4 vs. duplex DNA, and stabilize G-quadruplex structures. Two ligands described in this study include an acridine core which provides an aromatic surface capable of π-π interactions with the surface of G-quadruplexes. The successful synthesis of 4,5-diaminoacridine is described in chapter 2, as an essential fragment of the macrocycles (Mh) and (Mb). In order to investigate the synthetic method for macrocyclization, model compounds composing almost half of the designed macrocycles were explored. As discussed in chapter 3, the synthesis of the model compound for (Mb) turned out to be challenging. However, as a step towards the synthesis of (Mh), the synthesis of the hydrogen-containing model compound, which is almost half of the desired macrocycle (Mh) was achieved in our group and proved to be promising.

Intercepting Cyclic Dinucleotide Signaling with Small Molecules

Bacterial infections, especially the ones that are caused by multidrug-resistant strains, are becoming increasingly difficult to treat and put enormous stress on healthcare systems. Recently President Obama announced a new initiative to combat the growing problem of antibiotic resistance. New types of antibiotic drugs are always in need to catch up with the rapid speed of bacterial drug-resistance acquisition. Bacterial second messengers, cyclic dinucleotides, play important roles in signal transduction and therefore are currently generating great buzz in the microbiology community because it is believed that small molecules that inhibit cyclic dinucleotide signaling could become next-generation antibacterial agents. The first identified cyclic dinucleotide, c-di-GMP, has now been shown to regulate a large number of processes, such as virulence, biofilm formation, cell cycle, quorum sensing, etc. Recently, another cyclic dinucleotide, c-di-AMP, has emerged as a regulator of key processes in Gram-positive and mycobacteria. C-di-AMP is now known to regulate DNA damage sensing, fatty acid synthesis, potassium ion transport, cell wall homeostasis and host type I interferon response induction. Due to the central roles that cyclic dinucleotides play in bacteria, we are interested in small molecules that intercept cyclic dinucleotide signaling with the hope that these molecules would help us learn more details about cyclic dinucleotide signaling or could be used to inhibit bacterial viability or virulence. This dissertation documents the development of several small molecule inhibitors of a cyclic dinucleotide synthase (DisA from B. subtilis) and phosphodiesterases (RocR from P. aeruginosa and CdnP from M. tuberculosis). We also demonstrate that an inhibitor of RocR PDE can inhibit bacterial swarming motility, which is a virulence factor.

Helicase binding to DnaI exposes a cryptic DNA-binding site during helicase loading in Bacillus subtilis

The Bacillus subtilis DnaI, DnaB and DnaD proteins load the replicative ring helicase DnaC onto DNA during priming of DNA replication. Here we show that DnaI consists of a C-terminal domain (Cd) with ATPase and DNA-binding activities and an N-terminal domain (Nd) that interacts with the replicative ring helicase. A Zn2+-binding module mediates the interaction with the helicase and C67, C70 and H84 are involved in the coordination of the Zn2+. DnaI binds ATP and exhibits ATPase activity that is not stimulated by ssDNA, because the DNA-binding site on Cd is masked by Nd. The ATPase activity resides on the Cd domain and when detached from the Nd domain, it becomes sensitive to stimulation by ssDNA because its cryptic DNA-binding site is exposed. Therefore, Nd acts as a molecular 'switch' regulating access to the ssDNA binding site on Cd, in response to binding of the helicase. DnaI is sufficient to load the replicative helicase from a complex with six DnaI molecules, so there is no requirement for a dual helicase loader system.

Structural and functional interaction between polyamine related molecules and biological membranes

Changes induced by PA on nucleic acid (NA) conformation and synthesis is proven to be a major reason for PA essentiality (1-3). However, PA interactions with other polyanions, for instance polyanionic membrane lipid bilayers and glyosaminoglycans have received less attention (3-4). The functional importance of these interactions still is an obscure but interesting area of cell and molecular biology, especially in mammalian cells for which specific PA transport systems are not fully characterized (5). In mammals, activity and turnover of the polyamine (PA) synthesis key enzyme is controlled by a set of proteins: Antizymes (OAZ1-3) and antizyme inhibitors (AZIN1 and 2). It is demonstrated that AOZ modulate polyamine uptake (6), and that PA transport to mitochondria is linked to the respiratory chain state and modulates mitochondrial permeability transition (7). Antizyme expression variants have been located in mitochondria, being proposed as a proapoptotic factor (7-8). AZIN 2 is only expressed in a reduced set of tissues that includes mast cells, where it is associated to mast cell granules membrane (9). This fact, together to the abnormalities observed in bone marrow derived mast cell granules when they are differentiated under restricted PA synthesis conditions (10 and unpublished results), point out to important roles of PA and their related proteins in structure and function of mast cell granules. We will also present novel biophysical results on tripartite interactions of PA that remark the interest of the characterization of PA interactions with lipid bilayers for biomedicine and biotechnology. Thus, the information reported in this paper integrates previously reported information with our still unpublished results, all indicating that PA and their related proteins also are important factors for structure and dynamics of biological membranes and their associated functions essential in human physiology; for instance, solute interchange with the environment (uptake and secretion), oxidative metabolism and apoptosis. The importance of these involved processes for human homeostasis claim for further research efforts. 1. Ruiz-Chica J, Medina MA, Sánchez-Jiménez F and Ramírez FJ (2001) Fourier Transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines. Biophysical J. 80:443-454. 2. Lightfoot HL, Hall J (2014) Endogenous polyamine function--the RNA perspective. Nucleic Acids Res. 42:11275-11290. 3. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol. 42:39-51. 4. Finger S, Schwieger C, Arouri A, Kerth A, Blume A (2014) Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance. Biol Chem. 395:769-778. 5. Poulin R, Casero RA, Soulet D. (2012) Recent advances in the molecular biology of metazoan polyamine transport. Amino Acids. 42:711-723. 6. Kahana C (2009) Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor. Essays Biochem. 4:47-61. 7. Agostinelli E, Marques MP, Calheiros R, Gil FP, Tempera G, Viceconte N, Battaglia V, Grancara S, Toninello A (2010) Polyamines: fundamental characters in chemistry and biology. Amino Acids 38:393-403. 8. Liu GY, Liao YF, Hsu PC, Chang WH, Hsieh MC, Lin CY, Hour TC, Kao MC, Tsay GJ, Hung HC (2006) Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases' cascade. Apoptosis 11:1773-1788. 9. Kanerva K, Lappalainen J, Mäkitie LT, Virolainen S, Kovanen PT, Andersson LC (2009). Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion. PLoS One 31:e6858. 10. García-Faroldi G, Rodríguez CE, Urdiales JL, Pérez-Pomares JM, Dávila JC, Pejler G, Sánchez-Jiménez F, Fajardo I (2010) Polyamines are present in mast cell secretory granules and are important for granule homeostasis. PLoS One 30:e15071.

R-loops and G4-structures: from DNA damage to innate immune response gene activation

Non-B DNA structures like R-loops and G-quadruplexes play a pivotal role in several cellular vital processes like DNA transcription regulation. Misregulation of said non-canonical DNA structures can often lead to genome instability, DNA damage, and, eventually, to the activation of an innate immune response. For such reasons they have been studied as adjuvants in anticancer therapies. Here we studied drugs targeting R-loops (Top1 poisons) and G4s (hydrazone derivatives) in order to observe their effects in terms of DNA damage induction and, subsequently, activation of innate immune response. We studied how non-cytotoxic doses of ampthotecin and LMP-776 impact on genome instability, are capable to induce DNA damage and micronuclei, and, eventually lead to an innate immune gene response via the cGAS/STING pathway. G-quadruplexes are another ubiquitous, non-canonical DNA structure, more abundant in telomeric regions, demonstrating a marked relation with the impairment of telomerase and the regulation of DNA replication and transcription. Furthermore, we investigated the properties of new-synthesized molecules belonging to the highly promising class of hydrazone derivatives, in terms of cytotoxicity, ability to stabilize G4-structures, induce DNA damage, and activate interferon-B production. Both Top1 poisons and G4-stabilizers possess several features that can be very useful in clinical applications, in light of their ability to stimulate innate immune response factors and exert a certain cell-killing power, plus they offer a broad and diverse range of treatment options in order to face a variety of patient treatment needs. It is for these very reasons that it is of uttermost importance that further studies are conducted on these compounds, in order to synthesize new and increasingly powerful and flexible ones, with fewer side effects to customize therapies on specific cancers’ and patients’ features.

Dna Extraction From Filter-paper Spots Of Vaginal Samples Collected After Sexual Violence.

To detect the presence of male DNA in vaginal samples collected from survivors of sexual violence and stored on filter paper. A pilot study was conducted to evaluate 10 vaginal samples spotted on sterile filter paper: 6 collected at random in April 2009 and 4 in October 2010. Time between sexual assault and sample collection was 4-48hours. After drying at room temperature, the samples were placed in a sterile envelope and stored for 2-3years until processing. DNA extraction was confirmed by polymerase chain reaction for human β-globin, and the presence of prostate-specific antigen (PSA) was quantified. The presence of the Y chromosome was detected using primers for sequences in the TSPY (Y7/Y8 and DYS14) and SRY genes. β-Globin was detected in all 10 samples, while 2 samples were positive for PSA. Half of the samples amplified the Y7/Y8 and DYS14 sequences of the TSPY gene and 30% amplified the SRY gene sequence of the Y chromosome. Four male samples and 1 female sample served as controls. Filter-paper spots stored for periods of up to 3years proved adequate for preserving genetic material from vaginal samples collected following sexual violence.

Analysis Of The Dna Fourier Transform-infrared Microspectroscopic Signature Using An All-reflecting Objective.

The Fourier transform-infrared (FT-IR) signature of dry samples of DNA and DNA-polypeptide complexes, as studied by IR microspectroscopy using a diamond attenuated total reflection (ATR) objective, has revealed important discriminatory characteristics relative to the PO2(-) vibrational stretchings. However, DNA IR marks that provide information on the sample's richness in hydrogen bonds have not been resolved in the spectral profiles obtained with this objective. Here we investigated the performance of an all reflecting objective (ARO) for analysis of the FT-IR signal of hydrogen bonds in DNA samples differing in base richness types (salmon testis vs calf thymus). The results obtained using the ARO indicate prominent band peaks at the spectral region representative of the vibration of nitrogenous base hydrogen bonds and of NH and NH2 groups. The band areas at this spectral region differ in agreement with the DNA base richness type when using the ARO. A peak assigned to adenine was more evident in the AT-rich salmon DNA using either the ARO or the ATR objective. It is concluded that, for the discrimination of DNA IR hydrogen bond vibrations associated with varying base type proportions, the use of an ARO is recommended.

Prognostic Value Of Dna And Mrna E6/e7 Of Human Papillomavirus In The Evolution Of Cervical Intraepithelial Neoplasia Grade 2.

This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL). This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months. Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant. The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.

Phylogenetics, Ancestral State Reconstruction, And A New Infrafamilial Classification Of The Pantropical Ochnaceae (medusagynaceae, Ochnaceae S.str., Quiinaceae) Based On Five Dna Regions.

Ochnaceae s.str. (Malpighiales) are a pantropical family of about 500 species and 27 genera of almost exclusively woody plants. Infrafamilial classification and relationships have been controversial partially due to the lack of a robust phylogenetic framework. Including all genera except Indosinia and Perissocarpa and DNA sequence data for five DNA regions (ITS, matK, ndhF, rbcL, trnL-F), we provide for the first time a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogenetic backbone of the family. Based on this, we present a new classification of Ochnaceae s.l., with Medusagynoideae and Quiinoideae included as subfamilies and the former subfamilies Ochnoideae and Sauvagesioideae recognized at the rank of tribe. Our data support a monophyletic Ochneae, but Sauvagesieae in the traditional circumscription is paraphyletic because Testulea emerges as sister to the rest of Ochnoideae, and the next clade shows Luxemburgia+Philacra as sister group to the remaining Ochnoideae. To avoid paraphyly, we classify Luxemburgieae and Testuleeae as new tribes. The African genus Lophira, which has switched between subfamilies (here tribes) in past classifications, emerges as sister to all other Ochneae. Thus, endosperm-free seeds and ovules with partly to completely united integuments (resulting in an apparently single integument) are characters that unite all members of that tribe. The relationships within its largest clade, Ochnineae (former Ochneae), are poorly resolved, but former Ochninae (Brackenridgea, Ochna) are polyphyletic. Within Sauvagesieae, the genus Sauvagesia in its broad circumscription is polyphyletic as Sauvagesia serrata is sister to a clade of Adenarake, Sauvagesia spp., and three other genera. Within Quiinoideae, in contrast to former phylogenetic hypotheses, Lacunaria and Touroulia form a clade that is sister to Quiina. Bayesian ancestral state reconstructions showed that zygomorphic flowers with adaptations to buzz-pollination (poricidal anthers), a syncarpous gynoecium (a near-apocarpous gynoecium evolved independently in Quiinoideae and Ochninae), numerous ovules, septicidal capsules, and winged seeds with endosperm are the ancestral condition in Ochnoideae. Although in some lineages poricidal anthers were lost secondarily, the evolution of poricidal superstructures secured the maintenance of buzz-pollination in some of these genera, indicating a strong selective pressure on keeping that specialized pollination system.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

A Modified Acidic Approach For Dna Extraction From Plant Species Containing High Levels Of Secondary Metabolites.

Purified genomic DNA can be difficult to obtain from some plant species because of the presence of impurities such as polysaccharides, which are often co-extracted with DNA. In this study, we developed a fast, simple, and low-cost protocol for extracting DNA from plants containing high levels of secondary metabolites. This protocol does not require the use of volatile toxic reagents such as mercaptoethanol, chloroform, or phenol and allows the extraction of high-quality DNA from wild and cultivated tropical species.

Crosstalk Between Kinases, Phosphatases And Mirnas In Cancer.

Reversible phosphorylation of proteins, performed by kinases and phosphatases, is the major post translational protein modification in eukaryotic cells. This intracellular event represents a critical regulatory mechanism of several signaling pathways and can be related to a vast array of diseases, including cancer. Cancer research has produced increasing evidence that kinase and phosphatase activity can be compromised by mutations and also by miRNA silencing, performed by small non-coding and endogenously produced RNA molecules that lead to translational repression. miRNAs are believed to target about one-third of human mRNAs while a single miRNA may target about 200 transcripts simultaneously. Regulation of the phosphorylation balance by miRNAs has been a topic of intense research over the last years, spanning topics going as far as cancer aggressiveness and chemotherapy resistance. By addressing recent studies that have shown miRNA expression patterns as phenotypic signatures of cancers and how miRNA influence cellular processes such as apoptosis, cell cycle control, angiogenesis, inflammation and DNA repair, we discuss how kinases, phosphatases and miRNAs cooperatively act in cancer biology.

Elevated Micronucleus Frequency In Patients With Type 2 Diabetes, Dyslipidemia And Periodontitis.

The over-production of reactive oxygen species (ROS) can cause oxidative damage to a large number of molecules, including DNA, and has been associated with the pathogenesis of several disorders, such as diabetes mellitus (DM), dyslipidemia and periodontitis (PD). We hypothesise that the presence of these diseases could proportionally increase the DNA damage. The aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM, dyslipidemia and PD. One hundred and fifty patients were divided into five groups based upon diabetic, dyslipidemic and periodontal status (Group 1 - poor controlled DM with dyslipidemia and PD; Group 2 - well-controlled DM with dyslipidemia and PD; Group 3 - without DM with dyslipidemia and PD; Group 4 - without DM, without dyslipidemia and with PD; and Group 5 - without DM, dyslipidemia and PD). Blood analyses were carried out for fasting plasma glucose, HbA1c and lipid profile. Periodontal examinations were performed, and venous blood was collected and processed for micronucleus (MN) assay. The frequency of micronuclei was evaluated by cell culture cytokinesis-block MN assay. The general characteristics of each group were described by the mean and standard deviation and the data were submitted to the Mann-Whitney, Kruskal-Wallis, Multiple Logistic Regression and Spearman tests. The Groups 1, 2 and 3 were similarly dyslipidemic presenting increased levels of total cholesterol, low density lipoprotein cholesterol and triglycerides. Periodontal tissue destruction and local inflammation were significantly more severe in diabetics, particularly in Group 1. Frequency of bi-nucleated cells with MN and MNF, as well as nucleoplasmic bridges, were significantly higher for poor controlled diabetics with dyslipidemia and PD in comparison with those systemically healthy, even after adjusting for age, and considering Bonferroni's correction. Elevated frequency of micronuclei was found in patients affected by type 2 diabetes, dyslipidemia and PD. This result suggests that these three pathologies occurring simultaneously promote an additional role to produce DNA impairment. In addition, the micronuclei assay was useful as a biomarker for DNA damage in individuals with chronic degenerative diseases.