*2.4. Comparison of the Ca2*+*-Signaling in B and T Cells of Nkx2-3*−/− *and BALB*/*c Mice*

Since we found significant differences in the rhG1-induced immune responses of the Nkx2-3−/− and BALB/c mice, we were curious about what could be in the background of such variations. In the case of GIA, similarly to RA, close cooperation between T and B cells is necessary for the development of autoimmunity [7]. Since the Nkx2-3 mutation affects mostly the B lymphocyte development and recirculation, we hypothesized that the activation of B cells might be impaired, which, in turn, led to ameliorated arthritis. To characterize the activation of the B cells, we isolated the inguinal and mesenteric lymph nodes from Nkx2-3−/− or BALB/c control mice and loaded the lymphocytes with the Ca2+-specific indicator Fluo-3. Then, we activated the B cells with cross-linking of the BcR with anti-IgM or IgG antibodies and followed the intracellular Ca2+ signals (Figure 4).

We found significantly lower anti-IgM or anti-IgG-induced Ca2+-signal in the B cells isolated from the inguinal lymph nodes of Nkx2-3−/− mice than those from BALB/c (Figure 4A,C). Similarly, B cells isolated from the mesenteric lymph nodes, showed decreased Ca2+-signal both after anti-IgM or anti-IgG-activation (Figure 4B,D) when compared to BALB/c controls, however, these differences did not reach statistical significance. Finally, although we were not expecting any changes in the T cell activation in Nkx2-3−/− mice, we tested the Ca2+-signal of T cells after anti-CD3 cross-linking. As we expected, the Ca2+-signals of T cells isolated from both the inguinal and mesenteric lymph nodes were similar in Nkx2-3−/− and BALB/c mice indicating that, indeed, the Nkx2-3 mutation caused an activation perturbance specifically in B cells.

**Figure 4.** The comparison of the Ca2+-signals of B and T cells in Nkx2-3−/− and control BALB/c mice. Cells were isolated from the inguinal (**A**,**C**,**E**) or mesenteric (**B**,**D**,**F**) lymph nodes of Nkx2-3−/− and BALB/c mice and loaded with the Ca2+-specific indicator Fluo-3. Activation of B cells was induced by anti-IgM (**A**,**B**) or anti-IgG (**C**,**D**), activation of T cells was induced by anti-CD3 cross-linking (**E**,**F**). The changes in the intracellular Ca2+ levels of B or T cells were measured with a flow cytometer in the FL1 channel for five and a half minutes. Graphs show the time-dependent changes in the FL1 fluorescence (ratiometric with the intracellular Ca2+ level) as mean ± SEM values calculated from the data of *n* = 3 Nkx2-3−/− and *n* = 3 BALB/c mice. \* *p* < 0.05.

#### **3. Discussion**

In the present study, we set out to study how Nkx2-3-deficiency impacted the development of autoimmune arthritis. To answer this question, we used a mouse RA model, GIA, whereby we immunized Nkx2-3−/− and BALB/c control mice side-by-side with the rhG1 antigen. While the spleen plays important roles in the T-dependent immune responses [11,29], and in GIA it also serves as an activation niche for autoreactive lymphocytes [8,30], in Nkx2-3−/− mice, with severely damaged spleen, GIA could still be induced. This result shows that in GIA the spleen's role is not exclusive in the activation of autoreactive lymphocytes, and confirms that the lymph nodes and perhaps other lymphatic or extralymphatic tissues [8] may play an equally important role in the disease induction. On the other hand, we observed lower incidence and decreased arthritis severity in the Nkx2-3−/− mice compared to the wild-type controls, thus, the splenic defects had a significant impact on the rhG1-induced immune reaction. During the induction of (P)GIA, we immunized the mice with PG extracts/rhG1 and DDA intraperitoneally which led to the local activation of T lymphocytes both in the peritoneal cavity and the mesenteric lymph nodes followed by the systemic immune response in which the spleen is involved [8]. The local activation of Th1 and Th17 cells in the peritoneal cavity is of special importance because it was a specific feature of BALB/c mice which are the only susceptible mouse strain for (P)GIA [8]. The present results in Nkx2-3−/− mice might also support this: the local

peritoneal activation could remain unchanged, however, the systemic response, developing in the spleen is missing, which could be responsible for the weaker arthritis.

As it was previously described there is a significant B cell trafficking between the peritoneal cavity and the spleen [26]. Although there was a preferential homing of the peritoneal B1 and B2 cells towards other serosa surfaces like the pleura, however, some B1 and B2 cells migrated from the peritoneal cavity into the spleen [26]. Since in Nkx2-3−/− mice, the spleen microarchitecture is seriously defective, with abnormal adhesion molecule expression and vessel formation [29] most likely, the above-mentioned B cell trafficking is defective [29], in addition to the absence of MZ B cells involved in antigen delivery for GC initiation [21]. B lymphocytes not only play a role as precursors for antibody-producing plasma cells in autoimmune arthritis but have an equally important role as antigen-presenting cells [31]. Based on the above-mentioned works [26,29], we propose that the local, peritoneal antigen presentation by B cells could remain unchanged, however, due to the potential lack of splenic homing (and the severely reduced GC formation in the spleen upon T-dependent antigen challenge [21]), the systemic activation could be impaired. The subsequently weaker immune response is mirrored in the decreased splenic proliferation and cytokine production. This was particularly pronounced in the case of lymphocyte-derived cytokines (IL-4, IL-6, IL-17 and IFNγ), but, in the case of TNFα, we measured approximately equal amounts in Nkx2-3−/− and control BALB/c spleen cell cultures, showing that the macrophages were not affected.

In humans, the potential pathogenic role of the Nkx2-3 transcription factor was found in inflammatory bowel diseases (Crohn's and ulcerative colitis) so far [23,24] and also suggested in spondylarthritis more recently [32]. In mice, the absence of Nkx2-3 proved to be protective in DSS-induced colitis through an IL-22-independent mechanism [18]. To our knowledge, this was the first experiment where the Nkx2-3−/− mutation was investigated in the context of autoimmune arthritis. Based on the data presented in this study, Nkx2-3 is not only involved in intestinal inflammatory diseases, but also affects autoimmune arthritis.

An important finding of the present study was that not all Nkx2-3−/− mice developed arthritis upon rhG1 immunization: in all experiments, 20–40% of Nkx2-3−/− mice remained healthy contrary to BALB/c mice (>90% incidence, as seen here and in previous studies [5,27]). To decipher what could be the reason why certain immunized mice did not develop arthritis, we analyzed their immune response parameters separately. We measured considerably lower anti-rhG1 and anti-CCP IgG1 antibody levels in the sera of nonarthritic than in the sera of arthritic Nkx2-3−/− mice, respectively. This alone could explain the differences in the arthritis, since it has been shown in several earlier studies that the serum antibody levels against the proteoglycan aggrecan (and its immunodominant region: G1 domain) and CCP show the strongest correlation with the severity of GIA [33,34]. Furthermore, in nonarthritic Nkx2-3−/− mice, the serum concentrations of IL-1β, IL-4 and IL-6 were lower than in arthritic Nkx2-3−/− mice. We hypothesize that in those Nkx2-3−/− mice, which did not develop arthritis, the B cell activation and/or antigen presentation was inadequate to induce sufficient antibody production and autoreactive T cell activation. However, further and more detailed investigation would be needed to adequately answer this question.

Even in those Nkx2-3−/− mice which did develop GIA, there were significantly milder symptoms (lower clinical scores, lesser edema, radiologically decreased cartilage and bone destruction). This was in line with those serum parameters which were clearly different from the control BALB/c mice. Specifically, although the anti-rhG1 and anti-CCP-IgG1 antibody levels were similar in the arthritic Nkx2-3−/− mice and the controls, the anti-CCP-IgG2a antibodies were produced in lesser amounts. Additionally, the concentrations of signature cytokines of GIA [5], and likewise, RA [2], IFNγ and IL-17 were markedly lower, whereas the concentrations of IL-1β, IL-4 and IL-6 were markedly higher in Nkx2-3−/− than in BALB/c mice. Overall, these markers suggest a stronger Th2 activation (primarily indicated by IL-4 and anti-CCP IgG2a) in Nkx2-3−/− mice instead of the characteristic Th1/Th17 dominated immune response seen in GIA of BALB/c mice [5,8,28]. Since Th2 cytokines have primarily

anti-inflammatory effects [11] and have been shown to ameliorate PGIA [35], we suggest that this slight shift towards Th2 measured in Nkx2-3−/− mice might explain the milder arthritis.

As described here, and earlier [5], similarly to RA, in GIA mice significant anti-CCP antibody production can be detected. The process of citrullination and the role of autoantibodies against these modified antigens is of special interest in RA. ACPA production is promoted by environmental factors like smoking and genetic predispositions such as *HLA-DRB1* [36]. Several studies reported that ACPA positive patients are prone to have more joint erosions [36,37]. ACPA form immune complexes with the citrullinated peptides leading to the activation of macrophages and proinflammatory cytokine production, as well as osteoclastogenesis [36,37]. ACPA is highly specific to RA and can be early detected, even before the onset of RA. Therefore, it is used as a specific diagnostic marker for RA [38]. Recently, it was also found that the pathogenic ACPA are hyperglycosylated which might be regulated by Th17 cells [37]. Since in GIA Th17 activation was observed it is tempting to speculate that altered glycosylation might also occur which could contribute to the pathologic immune reaction against the cartilage antigen components.

Finally, to find a cellular mechanism in the background of the above-detailed immune response differences, we turned our attention to the activation of B cells. B cell activation starts with the engagement of the BcR by the antigen and followed by a well-characterized line of biochemical events including the phosphorylation of cytoplasmic signaling proteins and the transient elevation of the cytoplasmic Ca2+-level [11]. We studied the latter and found that in mesenteric and inguinal lymph node B cells of Nkx2-3−/− mice, in vitro stimulation with both anti-mouse IgM and anti-mouse IgG caused a weaker Ca2+-signal compared to BALB/c controls. At the same time, the Ca2+-signal in Nkx2-3−/− T cells remained unchanged, showing that this was a B-cell-specific alteration. This decreased B cell activation capacity of Nkx2-3−/− mice was also seen on phospho-blots from some preliminary experiments (data not shown), however, further analysis is needed to clarify which protein(s) could be involved in the signaling changes. These data are in harmony with those described earlier: increased expression of Nkx2-3 in B cells led to Syk and Lyn phosphorylation, elevated basal Ca2+-level and anti-IgM-induced Ca2+-signal [25], the exact opposite to what we found here, in the absence of Nkx2-3. The decreased antigen-driven B cell activation could lead to weaker B cell proliferation and differentiation which, in turn, could explain the less pronounced immune response and the consequently milder autoimmune arthritis.

In conclusion, the complex immune response changes in Nkx2-3−/− mice due to the defective spleen structure and function could explain the reduced severity and lower incidence of autoimmune arthritis. At the cellular level, we found weaker B cell activation which might, at least in part, be responsible for the altered immune response. These data add to our knowledge about the significance of the spleen in the development of autoimmunity, and hopefully serve as a starting point for future studies.


*2.1.8. The social rank theory of auditory hallucinations*

The social rank theory was generally used for depression and anxiety disorders but considering the parallel mechanisms within the scope of "attack the weaker and submit to the stronger," it was finally modified for hallucinations. Different from other cognitive theories, this theory considers *the patient's relationship with voices as well as with his significant others.* This approach uses the ABC framework. ABC model for auditory hallucinations of psychosis can be summarized as follows:

Activating events can be categorized into three types including symptoms and internal events (e.g., hallucinations), descriptions of interactions with significant others like parents or siblings, and significant life events (diagnosis, hospitalization, and social stigma). According to this theory, the hallucinations demonstrate *a core self-perception of low social rank*, so the person perceives that he/she is in control of his/her parents or peers and community. The emotional consequences of these evaluations can be shame, humiliation, and depression. In this context, the distress and behavior are related to patients' perceived relationship with voices, their appraisal of voices power and omnipotence, as a result they evaluate the voice as benevolent or malevolent [33, 36–38].

The explanations mentioned earlier would help to understand the occurrence of psychotic episodes. The following passages will also address the maintenance of these psychotic symptoms.


*Integrins in Ovarian Cancer: Survival Pathways, Malignant Ascites and Targeted Photochemistry DOI: http://dx.doi.org/10.5772/intechopen.106725*

potent inhibitors and found that JQ1, an inhibitor of the Myc oncogenic network, synergized with VS-6063. Although the efficacy was dependent on the cell line, using VS-6063 and JQ1 caused an additive or synergistic inhibition effect in proliferation and viability of ovarian cancer cells by inhibiting active FAK and c-Myc, as well as their signaling, through the PI3K/Akt pathway.


[24] Fogli L.G. Lisi E. Marrone A. Montanino D. Palazzo A. and Rotunno M.A. arXiv:

[26] Akhmedov K.E. Frigerio M. and Smirnov Y.A. *J. High Energy Phys.*, 09:021, 2003 [hep-

[29] Antusch S. Kersten J. Lindner M. and Ratz M. *Nucl. Phys. B*, 674:401, 2003 [hep-ph/

[30] Jeannerot R. Khalil S. Lazarides G. and Shafi Q. *J. High Energy Phys.*, 10:012, 2000 [hep-

[32] Gogoladze I. Khalid R. Raza S. and Shafi Q. *J. High Energy Phys.*, 12:055, 2010 [arXiv:

[34] Gómez E.M. Lazarides G. and Pallis C. *Nucl. Phys. B*, 638:165, 2002 [hep-ph/0203131].

[37] Pallis C. and Toumbas N. *J. Cosmol. Astropart. Phys.*, 02:019, 2011 [arXiv:1101.0325].

[40] Kofman L. Linde A.D. and Starobinsky A.D. *Phys. Rev. Lett.*, 73:3195, 1994 [hep-th/

*Universe (O. Sawada and A. Sugamoto, eds.), Tsukuba, Japan*, page 95, 1979.

*North Holland, Amsterdam*, page 315, 1979.

[25] Branco C.G. *et al*. *Nucl. Phys. B*, 640:202, 2002.

[35] Lazarides G. and Shafi Q. *Phys. Rev. D* , 58:071702.

[36] Dimopoulos K. *et al*. *J. High Energy Phys.*, 05:057, 2003.

[23] Tortola M. Valle F.W.J. and Vanegas D. arXiv:1205.4018.

[27] ¸Seno ˘guz V.N. *Phys. Rev. D* , 76:013005, 2007 [arXiv:0704.3048].

[28] Pallis C. and Shafi Q. *Phys. Rev. D* , 86:023523, 2012 [arXiv:1204.0252].

[31] King F.S. and Oliveira M. *Phys. Rev. D* , 63:015010, 2001 [hep-ph/0008183].

[33] King F.S. and Oliveira M. *Phys. Rev. D* , 63:095004, 2001 [hep-ph/0009287].

[38] Lyth H.D. and Riotto A. *Phys. Rept.*, 314:1, 1999 [hep-ph/9807278].

[39] Lazarides G. *J. Phys. Conf. Ser.*, 53:528, 2006 [hep-ph/0607032].

*Levy et al.. eds.)*, page 687, 1980.

**Chapter 11**

**Provisional chapter**

**Light Cold Dark Matter in a Two-Singlet Model**

**Light Cold Dark Matter in a Two-Singlet Model**

Understanding the nature of dark matter (DM) as well as the origin of baryon asymmetry are two of the most important questions in both Cosmology and Particle Physics. The failure of the Standard Model (SM) of the electroweak and strong interactions in accommodating such questions motivates the search for an explanation in the realm of new physics beyond it. The most attractive strategy so far has been to look into extensions of the SM that incorporate electrically neutral and colorless weakly interacting massive particles (WIMPs), with masses from one to a few hundred GeV, coupling constants in the milli-weak scale and lifetimes

The most popular extension of the SM is the minimal supersymmetric standard model (MSSM) in which the neutral lightest supersymmetric particle (LSP) is seen as a candidate for dark matter. Indeed, neutralinos are odd under *R* -parity and are only produced or destroyed in pairs, thus making the LSP stable [1]. They can annihilate through a t-channel sfermion exchange into Standard Model fermions, or via a t-channel chargino-mediated process into *W*+*W*−, or through an s-channel pseudoscalar Higgs exchange into fermion pairs. They can also undergo elastic scattering with nuclei through mainly a scalar Higgs exchange [2].

However, most particularly in light of the recent signal reported by CoGeNT [3], which favors a light dark matter (LDM) with a mass in the range 7 − 9GeV and nucleon scattering cross section *σ*det ∼ 10−4 pb, having a neutralino as a LDM candidate can be challenging.  This choice of parameters leads to a sizable contribution to the branching ratios of some rare decays, which then disfavors the scenario of light neutralinos [5]. Also, in the next-to-minimal supersymmetric standard model (NMSSM) with 12 input parameters [6], realizing a LDM with an elastic scattering cross section capable of generating the CoGeNT signal is possible only in a finely-tuned region of the parameters where the neutralino is mostly singlino and the light CP-even Higgs is singlet-like with a mass below a few GeV. In such a situation, it is very difficult to detect such a light Higgs at the collider. It

and reproduction in any medium, provided the original work is properly cited.

©2012 Abada and Nasri, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Abada and Nasri; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

is clear then that other alternative scenarios for LDM are needed [7].

Abdessamad Abada and Salah Nasri

Abdessamad Abada and Salah Nasri

http://dx.doi.org/10.5772/52243

longer than the age of the Universe.

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**


**5.2. Epidemiologic evidence**

There is epidemiologic evidence that exposure to antibiotics in early life is associated with increased risk of excess adiposity. Recently, epidemiological studies have shown that this phenomenon can also occur in humans starting in the fetal stage of life. In that sense, Mueller et al. observed in a U.S. cohort that the administration of antibiotics to women in the last two trimesters of pregnancy increased 84% the risk of obesity in children at 7 years old compared to children born to mothers without antibiotics administration at the same period [110]. Also, Mor et al. observed similar results in a study performed in Denmark where they showed that prenatal exposure to systemic antibacterials was associated with an increased risk of overweight and obesity at school age, and this association varies by birth weight [111].

After birth, the exposure to antibiotics has been associated to obesity due to the analysis of different human cohorts in various countries. In a Danish mother-child pairs cohort, Ajslev et al. showed antibiotic exposure in children during the first 6 months was associated with an increased risk of being overweight at 7 years of age; the effect was stronger in boys than in girls. In a U.K. cohort, Trasande et al. showed that antibiotic use in the first 6 months of life was associated with increased BMI at 10, 20, and 38 months of age [19]. Both studies also determined that maternal BMI was a contributing factor for the development of obesity following exposure to antibiotics in early life, with increased effects seen in children with mothers of normal weight compared with children from mothers who were overweight. Also, Azad et al. in a study of Canadian infants showed that antibiotics administered in the first year of life increased the likelihood of a child being overweight at 9 years and 12 years of age being almost seen in boys [112], which was consistent with the previous results from Ajslev et al. In a U.S. cohort, Bailey at al. observed that repeated exposure to broad-spectrum antibiotics at ages 0–23 months was associated with early childhood obesity. Importantly, this observation was associated with the use of broad-spectrum antibiotics, but not with the use of narrow-spectrum antibiotics.

Finally, in a multicenter, multicountry, cross-sectional study (The International Study of Asthma and Allergies in Childhood Phase III) Murphy et al. observed a significant interaction between sex and early-life antibiotic exposure. Exposure to antibiotics during the first 12 months of life was associated with a small increase in BMI in boys aged 5–8 years but not in girls in this large international cross-sectional survey.

Colonization of neonate's gut microbiota relies on vertical transmission from the mother at the time of delivery; thus, during pregnancy or early-life exposure to antibiotics could have effects on weight later in life by disturbing the proper establishment of the gut microbiota.

#### **5.3. Antibiotic exposure and dysbiosis in children**

Higher prescription rates were shown in preterm or term infants with relevant clinical problems. In a study involving neonates admitted to the neonatal intensive care unit in U.S. from 2005 to 2010, more than 88% of extremely low birth weight infants were administrated anti-

Over the last decade, several national and international health institutions have made an enormous effort to decrease antibiotic use in the pediatric population by educating parents about the futility of treating viral infections with antibiotics and about concerns of antibiotic resistance [94, 95]. But, despite a recent reduction, widespread antibiotic use in infants and children remains a relevant health problem in the entire industrialized world, mainly because

However, even in countries in which the prescribing pattern usually adheres to national guidelines with respect to the choice of antibiotics, antibiotics are still largely prescribed to

In addition to antibiotic exposure for infection prevention and therapy, children could potentially be substantially exposed to antibiotics through the food supply chain or, more rarely,

In last 50 years, farmers have been using low doses of antibiotics to promote growth and feed efficiency of pigs, cows, sheep, and poultry [102]. Different antibiotics have been demonstrated to have these effects independently of its class, chemical structure, and mode of action and spectrum of activity. Moreover, the effects on growth are greater when animals receive

Also, studies in mice using multiple types of antibiotics have further confirmed this association, as well as identifying early life as the key period for microbe-mediated programing of

Experiments with germ-free animal models have provided direct evidence of the key role of the microbiota in the association between low doses of antibiotics exposure and growth promotion. In 1963, Coates et al. showed that in germ-free chicken antibiotics alone have no growth promoting effects [108]. Recently, Cox et al. showed that germ-free mice who received the microbiota from mice treated with low dose penicillin gained more weight and fat mass

Then, there are two main findings from these experiments. First, early life is a critical time for metabolic development of the host, and second, the microbiome has a key role in this process and its disturbance duty to antibiotic exposure at this time affects the course of growth and

There is epidemiologic evidence that exposure to antibiotics in early life is associated with increased risk of excess adiposity. Recently, epidemiological studies have shown that

antibiotics early in life than if the exposure occurs later in life [103–105].

than mice colonized with microbiota from control animals [107].

most prescriptions were frequently inappropriate [96].

children, particularly to very young children [97–100].

biotics [93].

274 Adiposity - Omics and Molecular Understanding

drinking water [101].

**5.1. Evidence from animals**

host metabolism [106, 107].

development [109].

**5.2. Epidemiologic evidence**

Prospective studies have showed that changes in gut microbiota in early life may precede the development of overweight and obesity [113, 114].

In particular, some bacterial taxa has been associated with the risk of obesity development, regarding to this, a high abundance of intestinal *Bifidobacteria* in early life appears to be associated with lower risk of overweight [114, 115], whereas high amounts of *Bacteroides fragilis* increase the risk of obesity development [113]. Thus, likely factors that exert an impact on gut microbiota composition and functionality in early life may also modulate the risk of obesity development.

Therefore, antibiotic exposure during childhood can reduce the phylogenetic diversity and microbial load of the gut microbiota [116].

Regarding preterm infants it has been shown that treatment with amoxicillin and gentamicin during the first week of life reduced the bacterial diversity and raised the relative abundance of *Enterobacter* in the second and third weeks of life compared to preterm infants not exposed to antibiotics [117].

Moreover, administration of penicillin, ampicillin, cephalexin, gentamicin, amikacin, erythromycin, vancomycin, clindamycin, and teichomycin to preterm infants has been associated with a decrease in the relative abundance of *Bifidobacteriaceae, bacilli*, and *Lactobacillales spp*., commonly linked with a healthy status and an increase in the presence of potentially pathogenic *Enterobacteriaceae* [117–119]. Besides short-term-effects, the dysbiosis produced by antibiotics administration in infants may produce long-term effects like the persistence of the risk of obesity development. It has been observed that 3 months after of antibiotics persists the microbiota disruption [120]. However, antibiotic administration to neonates has been linked to several critical clinical conditions in which modification of the microbiota composition is thought to play a relevant role, in diseases such as necrotizing enterocolitis and sepsis [121, 122].

Antibiotic treatments in early life can lead to long-term alterations in microbiota composition that result in changes to host metabolic functions, particularly during development, increasing the risk of obesity [109].


**1. Introduction**

There is increased evidence of the association of air pollution and deleterious health effects. Particulate matter (PM) has obtained more attention, especially the small size components (PM10, PM2.5, UFP -ultrafine particles-) that carry on their surface different organic and inorganic elements whose composition differ with local and regional variations [1].

PMs might be identified by source, e.g., primary or secondary; combustion products, traffic, or by particle's size (aerodynamic diameter) (PM10, PM2.5, UFP -ultrafine particles-). This former parameter is important because the larger the particle the shorter the time it remains suspended in the air and the lower the risk of it being inhaled. Also the smaller the particles the higher the chances of deleterious health effects [1].

### **1.1. Metals in particulate matter, air pollution and sources**

Particles toxic components are complex mixtures of solids or liquids with different character‐ istics (e.g., mass, number, size, shape, surface area, chemical composition, acidity, solubility). Chemical components may be located on the surface or inside the particle. Considering the source there are natural and anthropogenic emissions. Natural sources include sea salt, volcanic ash, pollens, fungal spores, soil particles, forest fires and wind-blow dust [2]. An‐

thropogenic sources consist of fossil fuel combustion products, industrial process, mining activities, wood stove burning and cigarette smoking. In urban areas the main source of PM are motor vehicles especially those derived from diesel fuel combustion.

One of the components adhered to the small particles are metals that come from impurities derived from fuel additives and brakes and tires attrition. Transition metals are generated by non-exhaust emissions, impurities in fuel additives, or metallurgic process. Iron (Fe), nickel (Ni), vanadium (V), chromium (Cr) copper (Cu) are considered because its potential to produce Reactive Oxygen Species (ROS) in biological systems [1]. Others such as zinc (Zn) may also exert toxic effects by other mechanisms besides ROS production.

Heavy metals such as cadmium, lead and mercury are some of more common air pollutants emitted by industrial activities, combustion, extraction and processing activities. The diagnosis of health effects for heavy metals might be difficult if there is no previous evidence of the exposure source, while acute exposures usually occur in the workplace and are more easily identified; signs and symptoms differ within metals because each one interacts with different targets such as: specific metal-binding proteins (metallothioneins, transferrin, ferritin, cerulo‐ plasmin) in cases such as Cd, Cu, Hg, Ag, Mn Zn, Al, and Be; membrane carrier-proteins (phosphate and sulphate-transporters), divalent cation-transporters, some examples are V, Cr, Mo, Se. Heavy metals share with transition metals the possibility to exert its toxic effects by the production of ROS [2-4].


**2.1. Basic principle of WDM**

Wavelength division multiplexing (WDM) is a technology used to combine or retrieve two or more optical signals of different optical center wavelengths in a fiber. It allows fiber capacity to be expanded in the frequency domain from one channel to more than 100 channels. This is accomplished by first converting standard, non – WDM optical signals to signals with unique WDM wavelengths that correspond to the available channel center wavelengths in the WDM multiplexer and demultiplexer. Typically, this is done by replacing non – WDM transceivers with the proper WDM channel transceivers. WDM channels are defined and labeled by their center wavelength or frequency and channel spacing. The WDM channel wavelength assignment is an industry standard defined in ITU – T documentation. Then the different WDM signal wavelengths are combined into one fiber by the WDM multiplexer. In the fiber, the individual signals propagate with little interaction assuming low signal power. For high powers, interchannel interaction can occur. Once the signals reach the fiber link end, the WDM demultiplexer separates the signals by their wavelengths, back to individual fibers that are connected to their respective equipment receivers. Optical receivers have a broad reception spectrum, which includes all of C (1530- 1565 nm) band. Many receivers can also receive signals with wavelengths down to O (1260- 1360) band "in [11]".

234 Optical Communication

Obviously that it is easier to achieve a larger channel's spectral efficiency if for optical signal modulation and coding some of novel modulation formats are used. This novel (or advanced) modulation formats provide narrower optical signals spectrum or multilevel encoding schemes that ensure more bits per one symbol than it is in traditional modulation formats, for example, on – off keying (OOK) with non – return to zero (NRZ) encoding format (NRZ – OOK) "in reference[10]". Maximal spectral efficiency, which can be obtained with traditional OOK modulation formats, is about 0.4 bit/s/Hz "in [2]". "It has been reported in ref. [7,8]" that using such novel modulation formats as quadrature amplitude modulation (16 – QAM particularly) and orthogonal frequency – division multiplexing(OFDM) together with polarization division multiplexing (PDM) technique it

Study object of this chapter is optimal mixed WDM system configuration that provides lowest in system's channels detected signals BER values. This developed mixed WDM system's model is offered for the future design of backbone optical networks and can be considered under the concept of next generation optical network (NGON). Chosen optical signal modulations formats and per channel bit rates, according to authors' thoughts, are the most appropriate and probable at this moment. It was concluded after careful evaluation of current state of optical telecommunication networks, their possible and the most likely

With the explosive growth in demand for capacity in national, regional, and metropolitan optical networks, high spectral efficiency wavelength division multiplexing transmission becomes essential. Current WDM optical transport systems are primarily based on 10 Gbit/s channels that are modulated with on – off – keying on a 50 GHz channel grid. Capacity upgrade of these systems calls for 40 Gbit/s wavelength channels to be carried in the same

Wavelength division multiplexing (WDM) is a technology used to combine or retrieve two or more optical signals of different optical center wavelengths in a fiber. It allows fiber capacity to be expanded in the frequency domain from one channel to more than 100 channels. This is accomplished by first converting standard, non – WDM optical signals to signals with unique WDM wavelengths that correspond to the available channel center wavelengths in the WDM multiplexer and demultiplexer. Typically, this is done by replacing non – WDM transceivers with the proper WDM channel transceivers. WDM channels are defined and labeled by their center wavelength or frequency and channel spacing. The WDM channel wavelength assignment is an industry standard defined in ITU – T documentation. Then the different WDM signal wavelengths are combined into one fiber by the WDM multiplexer. In the fiber, the individual signals propagate with little interaction assuming low signal power. For high powers, interchannel interaction can occur.

system "in [5,9]". To achieve this, several technical challenges need to be resolved.

can be achieved SE larger than 6 bit/s/Hz and even reaches 7 bit/s/Hz.

development strategy and trends in the future.

**2.1. Basic principle of WDM**

**2. Necessity of mixed WDM transmission system**

The process of combining and separating the different wavelengths in the fiber is the basis of WDM technology. As a rough analogy, a WDM demultiplexer can be thought of as a prism separating different colors of light from the incident white light ray. Each color of light can represent a unique WDM signal. The white light is the aggregate of all colors of light and represents all the WDM signals propagating in the fiber. It should be noted that WDM wavelength and WDM channel have different meanings. WDM wavelength refers to the center wavelength of an optical signal or a WDM channel. WDM channel refers to an optical signal communications path that is defined by a center wavelength and a spectral pass band. It can be full duplex, half duplex, or simplex. Most telecommunication systems require full duplex communication channels. All full duplex fiber communication channels require two optical signals, one in each direction, to work properly. For a two fiber full duplex WDM system, the two laser signals (one at both ends of the fiber link on different fibers) are assigned the same wavelength, which is referred to as the channel assignment. However, in cases where only one fiber is used for full duplex communications, both lasers will have different wavelength assignments and therefore communication channel uses two WDM channels. Typically, most WDM systems will use two fibers for a WDM link. Therefore, the WDM wavelength assignment is the same for both lasers for that channel and is referred to as the channel assignment.

The heart of any WDM system is the basic WDM (also referred to as passive WDM). The basic WDM only consist of optical filters without any electronics. Therefore, it is completely passive and highly reliable. The filters are designed to pass a selected light spectral range, referred to as a channel, with low loss and reject or reflect all other spectrum. A combination of these filters results in a multichannel WDM multiplexer or demultiplexer. At the transmit end, the multiplexer combines the unique wavelengths from each channel port into one common fiber and at the receive end the demultiplexer coupler separates the combined signals from the common fiber to their respective channel ports. For some systems, the multiplexer and demultiplexer units are the same and are interchangeable (universal units) "in [11]".

In recent years the dramatic increase of demand for transmission capacity is observed and to secure an appropriate quality of service (QoS) level telecommunications services providers must constantly and continuously develop their transmission systems in use "in references [4,12]". Currently, as a study object of many scientific works have been chosen and focused directly on the fibre total transmission capacity increases, and this can happen in a three different ways. The first one, the existing 10 Gbit/s Dense WDM system upgrade, but in fact it is the substitution of existing system with 40 Gbit/s DWDM system or faster, because the only 10 Gbit/s system components, which can be used in new 40 Gbit/s system, are fibre, boosters

and some external modulated lasers, but all transmitter and receiver electrical parts with bandpass filters must be changed to a new one. The second one, channels compaction by location them closer to each other using smaller channel spacing between them, in that way increasing the number of channel in available transmission frequency spectrum "in [2,15]". In this case, the total transmission capacity increment is achieved only because of increasing the number of channels, as the individual transmission rate in each channel remains unchanged. And the third way, total transmission capacity increment, using channel compaction with simultaneous increment of individual channel's transmission bit rate.

It is clear, that none of the proposed fibre's transmission capacity increment solution can be realized immediately, but it requires a certain amount of time and work, as any solution should be implemented gradually in several stages to avoid unnecessary problems.